Cationic oil-in-water emulsions

ABSTRACT

This invention generally relates to cationic oil-in-water emulsions that can be used to deliver negatively charged molecules, such as an RNA molecule. The emulsion particles comprise an oil core and a cationic lipid. The cationic lipid can interact with the negatively charged molecule thereby anchoring the molecule to the emulsion particles. The cationic emulsions described herein are particularly suitable for delivering nucleic acid molecules (such as an RNA molecule encoding an antigen) to cells and formulating nucleic acid-based vaccines.

RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2011/043108 filed on Jul. 6, 2011, which claims the benefit ofU.S. Provisional Application No. 61/361,892, filed on Jul. 6, 2010, theentire teachings of each of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Nucleic acid therapeutics have promise for treating diseases rangingfrom inherited disorders to acquired conditions such as cancer,infectious disorders (AIDS), heart disease, arthritis, andneurodegenerative disorders (e.g., Parkinson's and Alzheimer's). Notonly can functional genes be delivered to repair a genetic deficiency orinduce expression of exogenous gene products, but nucleic acid can alsobe delivered to inhibit endogenous gene expression to provide atherapeutic effect Inhibition of gene expression can be mediated by,e.g., antisense oligonucleotides, double-stranded RNAs (e.g., siRNAs,miRNAs), or ribozymes.

A key step for such therapy is to deliver nucleic acid molecules intocells in vivo. However, in vivo delivery of nucleic acid molecules, inparticular RNA molecules, faces a number of technical hurdles. First,due to cellular and serum nucleases, the half life of RNA injected invivo is only about 70 seconds (see, e.g., Kurreck, Eur. J. Bioch.270:1628-44 (2003)). Efforts have been made to increase stability ofinjected RNA by the use of chemical modifications; however, there areseveral instances where chemical alterations led to increased cytotoxiceffects or loss of or decreased function. In one specific example, cellswere intolerant to doses of an RNAi duplex in which every secondphosphate was replaced by phosphorothioate (Harborth, et al, AntisenseNucleic Acid Drug Rev. 13(2): 83-105 (2003)). As such, there is a needto develop delivery systems that can deliver sufficient amounts ofnucleic acid molecules (in particular RNA molecules) in vivo to elicit atherapeutic response, but that are not toxic to the host.

Nucleic acid based vaccines are an attractive approach to vaccination.For example, intramuscular (IM) immunization of plasmid DNA encoding forantigen can induce cellular and humoral immune responses and protectagainst challenge. DNA vaccines offer certain advantages overtraditional vaccines using protein antigens, or attenuated pathogens.For example, as compared to protein vaccines, DNA vaccines can be moreeffective in producing a properly folded antigen in its nativeconformation, and in generating a cellular immune response. DNA vaccinesalso do not have some of the safety problems associated with killed orattenuated pathogens. For example, a killed viral preparation maycontain residual live viruses, and an attenuated virus may mutate andrevert to a pathogenic phenotype.

Another limitation of nucleic acid based vaccines is that large doses ofnucleic acid are generally required to obtain potent immune responses innon-human primates and humans. Therefore, delivery systems and adjuvantsare required to enhance the potency of nucleic acid based vaccines.Various methods have been developed for introducing nucleic acidmolecules into cells, such as calcium phosphate transfection, polyprenetransfection, protoplast fusion, electroporation, microinjection andlipofection.

Cationic lipids have been widely formulated as liposomes to delivergenes into cells. However, even a small amount of serum (˜10%) candramatically reduce the transfection activity of liposome/DNA complexesbecause serum contains anionic materials. Recently, cationic lipidemulsion was developed to deliver DNA molecules into cells. See, e.g.,Kim, et al., International Journal of Pharmaceutics, 295, 35-45 (2005).

U.S. Pat. Nos. 6,753,015 and 6,855,492 describe a method of deliveringnucleic acid molecules to a vertebrate subject using cationicmicroparticles. The microparticles comprise a polymer, such as apoly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, apolyorthoester, a polyanhydride, and the like, and are formed usingcationic surfactants. Nucleic acid molecules are adsorbed on thesurfaces of the microparticles.

Kim et al. (Pharmaceutical Research, vol. 18, pages 54-60, 2001) andChung et al. (Journal of Controlled Release, volume 71, pages 339-350,2001) describe various oil-in-water emulsion formulations that are usedto enhance in vitro and in vivo transfection efficiency of DNAmolecules.

Ott et al. (Journal of Controlled Release, volume 79, pages 1-5, 2002)describes an approach involving a cationic sub-micron emulsion as adelivery system/adjuvant for DNA. The sub-micron emulsion approach isbased on MF59, a potent squalene in water adjuvant which has beenmanufactured at large scale and has been used in a commercially approvedproduct)(Fluad®). 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) wasused to facilitate intracellular delivery of plasmid DNA.

Although DNA-based vaccines hold great promise for prevention andtreatment of diseases, general concerns have been raised regarding theirsafety. The introduced DNA molecules could potentially integrate intothe host genome or, due to their distribution to various tissues, couldlead to undesirable sustained expression of antigens. In addition,certain DNA viruses have also been used as a vehicle to deliver DNAmolecules. Because of their infectious properties, such viruses achievea very high transfection rate. The viruses used are genetically modifiedin such a manner that no functional infectious particles are formed inthe transfected cell. Despite these precautions, however, it is notpossible to rule out the risk of uncontrolled propagation of theintroduced gene and viral genes, for example due to potentialrecombination events. This also entails the risk of the DNA beinginserted into an intact gene of the host cell's genome by e.g.recombination, with the consequence that this gene may be mutated andthus completely or partially inactivated or may give rise tomisinformation. In other words, synthesis of a gene product which isvital to the cell may be completely suppressed or, alternatively, amodified or incorrect gene product is expressed. In addition, it isgenerally difficult to scale up the manufacture and purification ofclinical-grade viral vectors.

One particular risk occurs if the DNA is integrated into a gene which isinvolved in the regulation of cell growth. In this case, the host cellmay become degenerate and lead to cancer or tumor formation.Furthermore, if the DNA introduced into the cell is to be expressed, itis necessary for the corresponding DNA vehicle to contain a strongpromoter, such as the viral CMV promoter. The integration of suchpromoters into the genome of the treated cell may result in unwantedalterations of the regulation of gene expression in the cell. Anotherrisk of using DNA as an agent to induce an immune response (e.g. as avaccine) is the induction of pathogenic anti-DNA antibodies in thepatient into whom the foreign DNA has been introduced, so bringing aboutan undesirable immune response.

RNA molecules encoding an antigen or a derivative thereof may also beused as vaccines. RNA vaccines offer certain advantages as compared toDNA vaccines. First, RNA cannot integrate into the host genome thusabolishing the risk of malignancies. Second, due to the rapiddegradation of RNA, expression of the foreign transgene is oftenshort-lived, avoiding uncontrolled long term expression of the antigen.Third, RNA molecules only need to be delivered to the cytoplasm toexpress the encoded antigen, whereas DNA molecules must permeate throughthe nuclear membrane.

Nonetheless, compared with DNA-based vaccines, relatively minorattention has been given to RNA-based vaccines. RNAs andoligonucleotides are hydrophilic, negatively charged molecules that arehighly susceptible to degradation by nucleases when administered as atherapeutic or vaccine. Additionally, RNAs and oligonucleotides are notactively transported into cells. See, e.g., Vajdy, M., et al., Mucosaladjuvants and delivery systems for protein-, DNA- and RNA-basedvaccines, Immunol Cell Biol, 2004. 82(6): p. 617-27.

Ying et al. (Nature Medicine, vol. 5, pages 823-827, 1999) describes aself-replicating RNA vaccine in which naked RNA encoding β-galactosidasewas delivered and the induction of CD8+ cells was reported.

Montana et al. (Bioconjugate Chem. 2007, 18, pages 302-308) describesusing cationic solid-lipid nanoparticles as RNA carriers for genetransfer. It was shown that solid-lipid nanoparticles protected the RNAmolecule from degradation, and the expression of reporter protein(fluorescein) was detected after microinjecting the RNA-particle complexinto sea urchin eggs.

WO 2010/009277 discloses Nano Lipid Peptide Particles (NLPPs) comprising(a) an amphipathic peptide, (b) a lipid, and (c) at least oneimmunogenic species. In certain embodiments, the NLPPs also incorporatea positively charged “capturing agent,” such as a cationic lipid. Thecapturing agent is used to anchor a negatively charged immunogenicspecies (e.g., a DNA molecule or an RNA molecule). Preparation of NLPPrequires amphipathic peptides, which are used to solubilize the lipidcomponent and to form nano-particles.

Therefore, there is a need to provide delivery systems for nucleic acidmolecules or other negatively charged molecules. The delivery systemsare useful for nucleic acid-based vaccines, in particular RNA-basedvaccines.

SUMMARY OF THE INVENTION

This invention generally relates to cationic oil-in-water emulsions thatcan be used to deliver negatively charged molecules, such as an RNAmolecule to cells. The emulsion particles comprise an oil core and acationic lipid. The cationic lipid can interact with the negativelycharged molecule thereby anchoring the molecule to the emulsionparticles. The cationic emulsions described herein are particularlysuitable for delivering nucleic acid molecules (such as an RNA moleculeencoding an antigen) to cells and formulating nucleic acid-basedvaccines.

In one aspect, the invention provides a composition comprising an RNAmolecule complexed with a particle of a cationic oil-in-water emulsion,wherein the particle comprises (a) an oil core that is in liquid phaseat 25° C., and (b) a cationic lipid. Preferably, the cationicoil-in-water emulsion particle is not a Nano Lipid Peptide Particle(NLPP). Preferably, the oil core is in liquid phase at 4° C. Optionally,the average diameter of the emulsion particles is from about 80 nm toabout 180 nm and the N/P of the emulsion is at least 4:1. Optionally,the emulsion is buffered (e.g., with a citrate buffer, a succinatebuffer, an acetate buffer etc.) and has a pH from about 6.0 to about8.0; preferably about 6.2 to about 6.8, and contains no more than 30 mMinorganic salt (e.g., NaCl). Optionally, the emulsion further comprisesa nonionic tonicifying agent, such as a sugar, sugar alcohol or acombination thereof, in a sufficient quantity to make the emulsionisotonic.

In certain embodiments, the cationic oil-in-water emulsion furthercomprises a surfactant, such as a nonionic surfactant. Exemplarynonionic surfactants include, e.g., SPAN85 (sorbtian trioleate), Tween80 (polysorbate 80; polyoxyethylenesorbitan monooleate), or acombination thereof. The cationic oil-in-water emulsion may comprisefrom about 0.01% to about 2.5% (v/v) surfactant. For example, thecationic oil-in-water emulsion may comprise about 0.08% (v/v) Tween 80,or alternatively, about 0.5% (v/v) Tween 80 and about 0.5% (v/v) SPAN85.A Polyethylene Glycol (PEG) or PEG-lipid, such as PEG₂₀₀₀PE, PEG₅₀₀₀PE,PEG₁₀₀₀DMG, PEG₂₀₀₀DMG, PEG₃₀₀₀DMG, or a combination thereof, may alsobe used.

The composition comprising an RNA molecule complexed with a particle ofa cationic oil-in-water emulsion may comprise from about 0.005% to about1.25% (v/v) surfactant. For example, the composition comprising theRNA-emulsion complex may comprise about 0.04% (v/v) Tween 80(polysorbate 80; polyoxyethylenesorbitan monooleate), or alternatively,about 0.25% (v/v) Tween 80 and about 0.25% (v/v) SPAN85 (sorbtiantrioleate).

In certain embodiments, the cationic oil-in-water emulsion furthercomprises a phospholipid. Exemplary phospholipid include,1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), or Eggphosphatidylcholine (egg PC). For example, the cationic oil-in-wateremulsion may comprise from about 0.1 mg/ml to about 20 mg/ml(preferably, from about 0.1 mg/ml to about 10 mg/ml) DOPE, oralternatively, from about 0.1 mg/ml to about 20 mg/ml (preferably, fromabout 0.1 mg/ml to about 10 mg/ml) DPyPE, or alternatively, from about0.1 mg/ml to about 20 mg/ml (preferably, from about 0.1 mg/ml to about10 mg/ml) egg PC.

The composition comprising an RNA molecule complexed with a particle ofa cationic oil-in-water emulsion may comprise from about 0.05 mg/ml toabout 10 mg/ml (preferably, from about 0.05 mg/ml to about 5 mg/ml)DOPE, or alternatively, from about 0.05 mg/ml to about 10 mg/ml(preferably, from about 0.05 mg/ml to about 5 mg/ml) DPyPE, oralternatively, from about 0.05 mg/ml to about 10 mg/ml (preferably, fromabout 0.05 mg/ml to about 5 mg/ml) egg PC.

In certain embodiments, the cationic oil-in-water emulsion furthercomprises a polymer or a surfactant in the aqueous phase of theemulsion. Exemplary polymers include poloxamers such as Pluronic® F127(Ethylene Oxide/Propylene Oxide Block Copolymer:H(OCH₂CH₂)_(x)(OCH₃CH(CH₃))_(y)(OCH₂CH₂)_(n)OH). For example, thecationic oil-in-water emulsion may comprise from about 0.05% to about20% (w/v) polymer, or from about 0.1% to about 10% (w/v) polymer, suchas 0.5% (w/v) or 1% (w/v) Pluronic® F127. The composition comprising anRNA molecule complexed with a particle of a cationic oil-in-wateremulsion may comprise from about 0.025% to about 10% (v/v) polymer, orfrom about 0.5% to about 5% (v/v) polymer, such as 0.25% (w/v), or 0.5%(w/v) Pluronic® F127.

The emulsions may comprise components that can promote particleformation, improve the complexation between the negatively chargedmolecules and the cationic particles, facilitate appropriatedecomplexation/release of the negatively charged molecules (such as anRNA molecule), increase the stability of the negatively charged molecule(e.g., to prevent degradation of an RNA molecule), or preventaggregation of the emulsion particles.

In certain embodiments, the oil core may comprise an oil that isselected from the following: Castor oil, Coconut oil, Corn oil,Cottonseed oil, Evening primrose oil, Fish oil, Jojoba oil, Lard oil,Linseed oil, Olive oil, Peanut oil, Safflower oil, Sesame oil, Soybeanoil, Squalene, Sunflower oil, Wheatgerm oil, Mineral oil, or acombination thereof. Preferrably, the oil is Soybean oil, Sunflower oil,Olive oil, Squalene, or a combination thereof. The cationic oil-in-wateremulsion may comprise from about 0.2% to about 20% (v/v) oil, preferablyabout 0.08% to about 5% oil, about 0.08% oil, about 4% to about 5% oil,about 4% oil, about 4.3% oil, or about 5% oil. The compositioncomprising an RNA molecule complexed with a particle of a cationicoil-in-water emulsion may comprise from about 0.1% to about 10% (v/v)oil, preferably, from about 2% to about 2.5% (v/v) oil.

In certain embodiments, the cationic lipid is selected from one of thefollowing: 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),313-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), Lipids E0001-E0118 orE0119-E0180 as disclosed in Table 6 (pages 112-139) of WO 2011/076807(incorporated herein by reference), or a combination thereof.Particularly preferred cationic lipids include DOTAP, DC Cholesterol,and DDA.

In certain embodiments, the cationic lipid is selected from one of thefollowing: 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),3β-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), Lipids E0001-E0118 orE0119-E0180 as disclosed in Table 6 (pages 112-139) of WO 2011/076807(incorporated herein by reference),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), or a combinationthereof. Particularly preferred cationic lipids include DOTAP, DCCholesterol, DDA, DOTMA, DOEPC, DSTAP, DODAC, DODAP, and DLinDMA.

In certain embodiments, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 3 mg/ml, preferably from about 0.8 mg/mlto about 1.6 mg/ml DOTAP.

The composition comprising an RNA molecule complexed with a particle ofa cationic oil-in-water emulsion may comprise from about 0.4 mg/ml toabout 1.5 mg/ml, preferably from about 0.4 mg/ml to about 0.8 mg/mlDOTAP. Optionally, the average diameter of the emulsion particles isfrom about 80 nm to about 180 nm and the N/P of the emulsion is at least4:1. Optionally, the composition is buffered (e.g., with a citratebuffer, a succinate buffer, an acetate buffer etc.) and has a pH fromabout 6.0 to about 8.0; and contains no more than 30 mM inorganic salt(e.g., NaCl). Optionally, the composition further comprises a nonionictonicifying agent, such as a sugar, sugar alcohol or a combinationthereof, in a sufficient quantity to make the composition isotonic.

In certain embodiments, the cationic oil-in-water emulsion comprisesfrom about 0.62 mg/ml to about 4.92 mg/ml DC Cholesterol.

The composition comprising an RNA molecule complexed with a particle ofa cationic oil-in-water emulsion may comprise from about 0.31 mg/ml toabout 2.46 mg/ml DC Cholesterol. Optionally, the average diameter of theemulsion particles is from about 80 nm to about 180 nm and the N/P ofthe emulsion is at least 4:1. Optionally, the composition is buffered(e.g., with a citrate buffer, a succinate buffer, an acetate bufferetc.) and has a pH from about 6.0 to about 8.0; preferably about 6.2 toabout 6.8, and contains no more than 30 mM inorganic salt (e.g., NaCl).Optionally, the composition further comprises a nonionic tonicifyingagent, such as a sugar, sugar alcohol or a combination thereof, in asufficient quantity to make the composition isotonic.

In certain embodiments, the cationic oil-in-water emulsion comprisesfrom about 0.73 mg/ml to about 1.45 mg/ml DDA.

The composition comprising an RNA molecule complexed with a particle ofa cationic oil-in-water emulsion may comprise from about 0.365 mg/ml toabout 0.725 mg/ml DDA. Optionally, the average diameter of the emulsionparticles is from about 80 nm to about 180 nm and the N/P of theemulsion is at least 4:1. Optionally, the composition is buffered (e.g.,with a citrate buffer, a succinate buffer, an acetate buffer etc.) andhas a pH from about 6.0 to about 8.0; preferably about 6.2 to about 6.8,and contains no more than 30 mM inorganic salt (e.g., NaCl). Optionally,the composition further comprises a nonionic tonicifying agent, such asa sugar, sugar alcohol or a combination thereof, in a sufficientquantity to make the composition isotonic.

In certain embodiments, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 3 mg/ml, preferably from about 0.8 mg/mlto about 1.6 mg/ml DOTMA.

The composition comprising an RNA molecule complexed with a particle ofa cationic oil-in-water emulsion may comprise from about 0.4 mg/ml toabout 1.5 mg/ml, preferably from about 0.4 mg/ml to about 0.8 mg/mlDOTMA. Optionally, the average diameter of the emulsion particles isfrom about 80 nm to about 180 nm and the N/P of the emulsion is at least4:1. Optionally, the composition is buffered (e.g., with a citratebuffer, a succinate buffer, an acetate buffer etc.) and has a pH fromabout 6.0 to about 8.0; preferably about 6.2 to about 6.8, and containsno more than 30 mM inorganic salt (e.g., NaCl). Optionally, thecomposition further comprises a nonionic tonicifying agent, such as asugar, sugar alcohol or a combination thereof, in a sufficient quantityto make the composition isotonic.

In certain embodiments, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 3 mg/ml, preferably from about 0.8 mg/mlto about 1.8 mg/ml DOEPC.

The composition comprising an RNA molecule complexed with a particle ofa cationic oil-in-water emulsion may comprise from about 0.4 mg/ml toabout 1.5 mg/ml, preferably from about 0.4 mg/ml to about 0.9 mg/mlDOEPC. Optionally, the average diameter of the emulsion particles isfrom about 80 nm to about 180 nm and the N/P of the emulsion is at least4:1. Optionally, the composition is buffered (e.g., with a citratebuffer, a succinate buffer, an acetate buffer etc.) and has a pH fromabout 6.0 to about 8.0; preferably about 6.2 to about 6.8, and containsno more than 30 mM inorganic salt (e.g., NaCl). Optionally, thecomposition further comprises a nonionic tonicifying agent, such as asugar, sugar alcohol or a combination thereof, in a sufficient quantityto make the composition isotonic.

In certain embodiments, the cationic oil-in-water emulsion comprisesfrom about 0.73 mg/ml to about 1.45 mg/ml DODAC.

The composition comprising an RNA molecule complexed with a particle ofa cationic oil-in-water emulsion may comprise from about 0.365 mg/ml toabout 0.725 mg/ml DODAC. Optionally, the average diameter of theemulsion particles is from about 80 nm to about 180 nm and the N/P ofthe emulsion is at least 4:1. Optionally, the composition is buffered(e.g., with a citrate buffer, a succinate buffer, an acetate bufferetc.) and has a pH from about 6.0 to about 8.0; preferably about 6.2 toabout 6.8, and contains no more than 30 mM inorganic salt (e.g., NaCl).Optionally, the composition further comprises a nonionic tonicifyingagent, such as a sugar, sugar alcohol or a combination thereof, in asufficient quantity to make the composition isotonic.

In one example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the cationic oil-in-water emulsioncomprises (a) about 0.5% (v/v) oil, and (b) a cationic lipid.

In one example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the composition comprises (a) about 0.25%(v/v) oil, and (b) a cationic lipid.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil core,(b) a cationic lipid, and (c) a phospholipid. Preferred phospholipidsinclude, e.g., DPyPE, DOPE, and egg PC. Preferably, the composition(negatively charged molecule-emulsion complex) comprises from about 0.05mg/ml to about 10 mg/ml (more preferably, from about 0.05 mg/ml to about5 mg/ml) DOPE, or alternatively, from about 0.05 mg/ml to about 10 mg/ml(more preferably, from about 0.05 mg/ml to about 5 mg/ml) DPyPE, oralternatively, from about 0.05 mg/ml to about 10 mg/ml (more preferably,from about 0.05 mg/ml to about 5 mg/ml) egg PC.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil coreand (b) DOTAP, and wherein the oil-in-water emulsion comprises fromabout 0.8 mg/ml to about 3.0 mg/ml DOTAP, preferably from about 0.8mg/ml to about 1.6 mg/ml DOTAP. In some embodiments, the negativelycharged molecule is RNA, the average diameter of the emulsion particlesis from about 80 nm to about 180 nm and the N/P of the emulsion is atleast 4:1. Optionally, the composition is buffered (e.g., with a citratebuffer, a succinate buffer, an acetate buffer etc.) and has a pH fromabout 6.0 to about 8.0; and contains no more than 30 mM inorganic salt(e.g., NaCl). Optionally, the composition further comprises a nonionictonicifying agent, such as a sugar, sugar alcohol or a combinationthereof, in a sufficient quantity to make the composition isotonic.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil coreand (b) DOTAP, and wherein the composition comprises from about 0.4mg/ml to about 1.5 mg/ml DOTAP, such as 0.4 mg/ml, 0.6 mg/ml, 0.7 mg/ml,0.8 mg/ml, etc. In some embodiments, the negatively charged molecule isRNA, the average diameter of the emulsion particles is from about 80 nmto about 180 nm and the N/P of the emulsion is at least 4:1. Optionally,the composition is buffered (e.g., with a citrate buffer, a succinatebuffer, an acetate buffer etc.) and has a pH from about 6.0 to about8.0; preferably about 6.2 to about 6.8, and contains no more than 30 mMinorganic salt (e.g., NaCl). Optionally, the composition furthercomprises a nonionic tonicifying agent, such as a sugar, sugar alcoholor a combination thereof, in a sufficient quantity to make thecomposition isotonic.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil coreand (b) DC Cholesterol, and wherein the oil-in-water emulsion comprisesfrom about 2.46 mg/ml to about 4.92 mg/ml DC Cholesterol. In someembodiments, the negatively charged molecule is RNA, the averagediameter of the emulsion particles is from about 80 nm to about 180 nmand the N/P of the emulsion is at least 4:1. Optionally, the compositionis buffered (e.g., with a citrate buffer, a succinate buffer, an acetatebuffer etc.) and has a pH from about 6.0 to about 8.0; preferably about6.2 to about 6.8, and contains no more than 30 mM inorganic salt (e.g.,NaCl). Optionally, the composition further comprises a nonionictonicifying agent, such as a sugar, sugar alcohol or a combinationthereof, in a sufficient quantity to make the composition isotonic.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil coreand (b) DC Cholesterol, and wherein the composition comprises from about1.23 mg/ml to about 2.46 mg/ml DC Cholesterol, such as 1.23 mg/ml. Insome embodiments, the negatively charged molecule is RNA, the averagediameter of the emulsion particles is from about 80 nm to about 180 nmand the N/P of the emulsion is at least 4:1. Optionally, the compositionis buffered (e.g., with a citrate buffer, a succinate buffer, an acetatebuffer etc.) and has a pH from about 6.0 to about 8.0, preferably about6.2 to about 6.8; and contains no more than 30 mM inorganic salt (e.g.,NaCl). Optionally, the composition further comprises a nonionictonicifying agent, such as a sugar, sugar alcohol or a combinationthereof, in a sufficient quantity to make the composition isotonic.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil coreand (b) DDA, and wherein the oil-in-water emulsion comprises from about0.73 mg/ml to about 1.45 mg/ml DDA. In some embodiments, the negativelycharged molecule is RNA, the average diameter of the emulsion particlesis from about 80 nm to about 180 nm and the N/P of the emulsion is atleast 4:1. Optionally, the composition is buffered (e.g., with a citratebuffer, a succinate buffer, an acetate buffer etc.) and has a pH fromabout 6.0 to about 8.0, preferably about 6.2 to about 6.8; and containsno more than 30 mM inorganic salt (e.g., NaCl). Optionally, thecomposition further comprises a nonionic tonicifying agent, such as asugar, sugar alcohol or a combination thereof, in a sufficient quantityto make the composition isotonic.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil coreand (b) DDA, and wherein the composition comprises from about 0.365mg/ml to about 0.725 mg/ml DDA, such as 0.725 mg/mL. In someembodiments, the negatively charged molecule is RNA, the averagediameter of the emulsion particles is from about 80 nm to about 180 nmand the N/P of the emulsion is at least 4:1. Optionally, the compositionis buffered (e.g., with a citrate buffer, a succinate buffer, an acetatebuffer etc.) and has a pH from about 6.0 to about 8.0, preferably about6.2 to about 6.8; and contains no more than 30 mM inorganic salt (e.g.,NaCl). Optionally, the composition further comprises a nonionictonicifying agent, such as a sugar, sugar alcohol or a combinationthereof, in a sufficient quantity to make the composition isotonic.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil coreand (b) DOTMA, and wherein the composition comprises from about 0.4mg/ml to about 1.5 mg/ml, preferably from about 0.4 mg/ml to about 0.8mg/ml DOTMA. In some embodiments, the negatively charged molecule isRNA, the average diameter of the emulsion particles is from about 80 nmto about 180 nm and the N/P of the emulsion is at least 4:1. Optionally,the composition is buffered (e.g., with a citrate buffer, a succinatebuffer, an acetate buffer etc.) and has a pH from about 6.0 to about8.0, preferably about 6.2 to about 6.8; and contains no more than 30 mMinorganic salt (e.g., NaCl). Optionally, the composition furthercomprises a nonionic tonicifying agent, such as a sugar, sugar alcoholor a combination thereof, in a sufficient quantity to make thecomposition isotonic.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil coreand (b) DOEPC, and wherein the composition comprises from about 0.4mg/ml to about 1.5 mg/ml, preferably from about 0.4 mg/ml to about 0.9mg/ml DOEPC. In some embodiments, the negatively charged molecule isRNA, the average diameter of the emulsion particles is from about 80 nmto about 180 nm and the N/P of the emulsion is at least 4:1. Optionally,the composition is buffered (e.g., with a citrate buffer, a succinatebuffer, an acetate buffer etc.) and has a pH from about 6.0 to about8.0, preferably about 6.2 to about 6.8; and contains no more than 30 mMinorganic salt (e.g., NaCl). Optionally, the composition furthercomprises a nonionic tonicifying agent, such as a sugar, sugar alcoholor a combination thereof, in a sufficient quantity to make thecomposition isotonic.

In another example, the invention provides a composition comprising anegatively charged molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil coreand (b) DODAC, and wherein the composition comprises from about 0.365mg/ml to about 0.725 mg/ml DODAC. In some embodiments, the negativelycharged molecule is RNA, the average diameter of the emulsion particlesis from about 80 nm to about 180 nm and the N/P of the emulsion is atleast 4:1. Optionally, the composition is buffered (e.g., with a citratebuffer, a succinate buffer, an acetate buffer etc.) and has a pH fromabout 6.0 to about 8.0, preferably about 6.2 to about 6.8; and containsno more than 30 mM inorganic salt (e.g., NaCl). Optionally, thecomposition further comprises a nonionic tonicifying agent, such as asugar, sugar alcohol or a combination thereof, in a sufficient quantityto make the composition isotonic.

Examples of negatively charged molecules include negatively chargedpeptide-containing antigens, nucleic acid molecules (e.g., RNA or DNA)that encode one or more peptide-containing antigens, negatively chargedsmall molecules, and negatively charged immunological adjuvants.Negatively charged immunological adjuvants include, e.g.,immunostimulatory oligonucleotides (e.g., CpG oligonucleotides),single-stranded RNAs, small molecule immune potentiators (SMIPs), etc.Negatively charged small molecules includes, e.g. phosphonate,fluorophosphonate, etc.

In certain embodiments, the negatively charged molecule is a nucleicacid molecule, such as an RNA molecule, that encodes an antigen. Incertain embodiments, the RNA molecule is a self-replicating RNAmolecule, such as an alphavirus-derived RNA replicon.

In another aspect, the invention provides immunogenic cationicoil-in-water emulsion comprising emulsion particles that contain an oilcore (preferably that is in liquid phase at 25° C.) and a cationiclipid, and a nucleic acid molecule that is complexed to the emulsionparticles, and wherein the average diameter of the emulsion particles isfrom about 80 nm to about 180 nm and the N/P of the emulsion is at least4:1. In certain embodiments, the nucleic acid molecule is an RNA, suchas a self replicating RNA. Preferably, the immunogenic cationicoil-in-water emulsion is buffered (e.g., with a citrate buffer, asuccinate buffer, an acetate buffer etc.) and has a pH from about 6.0 toabout 8.0, preferably about 6.2 to about 6.8; and contains no more than30 mM inorganic salt (e.g., NaCl). Preferably, the immunogenic cationicoil-in-water emulsion further comprises a nonionic tonicifying agent,such as a sugar, sugar alcohol or a combination thereof, in a sufficientquantity to make the emulsion isotonic.

In another aspect, the invention provides a method of preparing acomposition that comprises a negatively charged molecule complexed witha particle of a cationic oil-in-water emulsion, comprising: (A)preparing a cationic oil-in-water emulsion wherein the emulsioncomprises: (1) from about 0.2% to about 20% (v/v) oil, (2) from about0.01% to about 2.5% (v/v) surfactant, and (3) a cationic lipid that isselected from the group consisting of: (i) from about 0.8 mg/ml to about1.6 mg/ml DOTAP, (ii) from about 2.46 mg/ml to about 4.92 mg/ml DCCholesterol, and (iii) from about 0.73 mg/ml to about 1.45 mg/ml DDA;and (B) adding the negatively charged molecule to the cationicoil-in-water emulsion so that the negatively charged molecule complexeswith the particle of the emulsion.

In another aspect, the invention provides a method of preparing acomposition that comprises a negatively charged molecule complexed witha particle of a cationic oil-in-water emulsion, comprising: (A)preparing a cationic oil-in-water emulsion wherein the emulsioncomprises: (1) from about 0.2% to about 20% (v/v) oil, (2) from about0.01% to about 2.5% (v/v) surfactant, and (3) a cationic lipid that isselected from the group consisting of: (i) from about 0.8 mg/ml to about1.6 mg/ml DOTAP, (ii) from about 2.46 mg/ml to about 4.92 mg/ml DCCholesterol, (iii) from about 0.73 mg/ml to about 1.45 mg/ml DDA, (iv)from about 0.8 mg/ml to about 1.6 mg/ml DOTMA, (v) from about 0.8 mg/mlto about 1.8 mg/ml DOEPC; and (vi) from about 0.73 mg/ml to about 1.45mg/ml DODAC; and (B) adding the negatively charged molecule to thecationic oil-in-water emulsion so that the negatively charged moleculecomplexes with the particle of the emulsion.

In certain embodiments, the cationic oil-in-water emulsion is preparedby the process comprising: (1) combining the oil and the cationic lipidto form the oil phase of the emulsion; (2) providing the aqueous phase(i.e., continuous phase) of the emulsion; and (3) dispersing the oilphase in the aqueous phase by homogenization. The cationic lipid may bedissolved directly in the oil. Alternatively, the cationic lipid may bedissolved in any suitable solvent, such as chloroform (CHCl₃) ordichloromethane (DCM). Isopropyl alcohol may also be used. The solventmay be evaporated before the oil phase is added to the aqueous phase, orafter the oil phase is added to the aqueous phase but beforehomogenization. Alternatively, in instances where lipid solubility canbe an issue, a primary emulsion can be made with the solvent (e.g., DCM)still in the oil phase. In that case, the solvent would be allowed toevaporate directly from the emulsion prior to a secondaryhomogenization.

Additional optional steps to promote particle formation, to improve thecomplexation between the negatively charged molecules and the cationicparticles, to increase the stability of the negatively charged molecule(e.g., to prevent degradation of an RNA molecule), to facilitateappropriate decomplexation/release of the negatively charged molecules(such as an RNA molecule), or to prevent aggregation of the emulsionparticles may be included. For example, a polymer (e.g., Pluronic® F127)or a surfactant may be added to the aqueous phase of the emulsion. Inone exemplary embodiment, Pluronic® F127 is added to the RNA moleculeprior to complexation to the emulsion particles. Addition of Pluronic®F127 can increase the stability of the RNA molecule and further reduceRNA degradation. Poloxamer polymers can also promote the release of theRNA molecule and prevent aggregation of the emulsion particles. Finally,poloxamer polymers also have immune modulatory effect. See, e.g.,Westerink et al., Vaccine. 2001 Dec. 12; 20(5-6):711-23.

Preferably, the RNA molecule of the RNA-cationic particle complex ismore resistant to RNase degradation as compared to uncomplexed RNAmolecule.

In another aspect, the invention provides a pharmaceutical compositioncomprising a negatively charged molecule complexed with a particle of acationic oil-in-water emulsion, as described herein, and may furthercomprise one or more pharmaceutically acceptable carriers, diluents, orexcipients. In preferred embodiments, the pharmaceutical composition isa vaccine.

In another aspect, the invention provides a method of generating animmune response in a subject, comprising administering to a subject inneed thereof a composition as described herein.

The invention also relates to a pharmaceutical composition as describedherein for use in therapy, and to the use of a pharmaceuticalcomposition as described herein for the manufacture of a medicament forpotentiating or generating an immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stability of mouse thymus RNA in the presence of RNaseafter the RNA molecule was complexed with cationic nano-emulsion (CNE)particles. All samples were incubated with RNase for 30 minutes. RNasewas inactivated with proteinase K. Samples that were formulated withCNEs were decomplexed and analyzed for RNA integrity by denaturing gelelectrophoresis. Unlabeled lane contains molecular weight markers. Lanes1 and 2: mouse thymus RNA before (1) and after (2) RNase digestion;lanes 3 and 4: mouse thymus RNA complexed with CNE01 at an N/P ratio of10:1 before (3) and after (4) RNase digestion; lanes 5 and 6: mousethymus RNA complexed with CNE01 at an N/P ratio of 4:1 before (5) andafter (6) RNase digestion; lanes 7 and 8: mouse thymus RNA complexedwith CNE17 at an N/P ratio of 10:1 before (7) and after (8) RNasedigestion; lane 9 mouse thymus RNA complexed with CNE17 at an N/P ratioof 4:1 before (9) RNase digestion.

FIG. 2 shows the stability of mouse thymus RNA in the presence of RNaseafter the RNA molecule was complexed with CNE particles. All sampleswere incubated with RNase for 30 minutes. RNase was inactivated withproteinase K. Samples that were formulated with CNEs were decomplexedand analyzed for RNA integrity by denaturing gel electrophoresis. Theunlabeled lane contains molecular weight markers. Lane 10: mouse thymusRNA complexed with CNE17 at an N/P ratio of 4:1 after (10) RNasedigestion; lanes 11 and 12: mouse thymus RNA before (11) and after (12)RNase digestion; lanes 13 and 14: mouse thymus RNA complexed with CNE12at an N/P ratio of 10:1 before (13) and after (14) RNase digestion;lanes 15 and 16: mouse thymus RNA complexed with CNE12 at an N/P ratioof 4:1 before (15) and after (16) RNase digestion; lanes 17 and 18:mouse thymus RNA complexed with CNE13 at an N/P ratio of 10:1 before(17) and after (18) RNase digestion; lanes 19 and 20 mouse:thymus RNAcomplexed with CNE13 at an N/P ratio of 4:1 before (19) and after (20)RNase digestion.

FIG. 3 shows the stability of mouse thymus RNA in the presence of RNaseafter the RNA molecule was complexed with CNE particles. All sampleswere incubated with RNase for 30 minutes. RNase was inactivated withproteinase K. Samples that were formulated with CNEs were decomplexedand analyzed for RNA integrity by denaturing gel electrophoresis.Unlabeled lane contains molecular weight markers. Lanes 1 and 2: mousethymus RNA before (1) and after (2) RNase digestion; lanes 3 and 4mouse: thymus RNA complexed with CNE01 at an N/P ratio of 10:1 before(3) and after (4) RNase digestion; lanes 5 and 6: mouse thymus RNAcomplexed with CNE01 at an N/P ratio of 4:1 before (5) and after (6)RNase digestion; lanes 7 and 8: mouse thymus RNA complexed with CNE02 atan N/P ratio of 10:1 before (7) and after (8) RNase digestion; lane 9:mouse thymus RNA complexed with CNE02 at an N/P ratio of 4:1 before (9)RNase digestion.

FIG. 4 shows the stability of mouse thymus RNA in the presence of RNaseafter the RNA molecule was complexed with CNE particles. All sampleswere incubated with RNase for 30 minutes. RNase was inactivated withproteinase K and samples that were formulated were decomplexed andanalyzed for RNA integrity by denaturing gel electrophoresis. Unlabeledlane contains molecular weight markers. Lanes 15 and 16: mouse thymusRNA before (15) and after (16) RNase digestion; lanes 17 and 18: mousethymus RNA complexed with CNE04 at an N/P ratio of 10:1 before (17) andafter (18) RNase digestion; lanes 19 and 20: mouse thymus RNA complexedwith CNE04 at an N/P ratio of 4:1 before (19) and after (20) RNasedigestion; lanes 21 and 22: mouse thymus RNA complexed with CNE05 at anN/P ratio of 10:1 before (21) and after (22) RNase digestion; lanes 23and 24: mouse thymus RNA complexed with CNE05 at an N/P ratio of 4:1before (23) and after (24) RNase digestion.

FIG. 5 shows the stability of mouse thymus RNA in the presence of RNaseafter the RNA molecule was complexed with CNE particles. All sampleswere incubated with RNase for 30 minutes. RNase was inactivated withproteinase K and samples that were formulated were decomplexed andanalyzed for RNA integrity by denaturing gel electrophoresis. Unlabeledlanes contain molecular weight markers. Lanes 1 and 2: mouse thymus RNAbefore (1) and after (2) RNase digestion; lanes 3 and 4: mouse thymusRNA complexed with CNE17 at an N/P ratio of 10:1 before (3) and after(4) RNase digestion; lanes 5 and 6: mouse thymus RNA complexed withCNE17 at an N/P ratio of 4:1 before (5) and after (6) RNase digestion;lanes 7 and 8: with CNE27 at an N/P ratio of 10:1 before (7) and after(8) RNase digestion; lanes 9 and 10: mouse thymus RNA complexed withCNE27 at an N/P ratio of 4:1 before (9) and after (10) RNase digestion;lanes 11 and 12: mouse thymus RNA before (11) and after (12) RNasedigestion; lanes 13 and 14: mouse thymus RNA complexed with CNE32 at anN/P ratio of 10:1 before (13) and after (14) RNase digestion; lanes 15and 16: mouse thymus RNA complexed with CNE32 at an N/P ratio of 4:1before (15) and after (16) RNase digestion.

FIG. 6 shows the stability of mouse thymus RNA in the presence of RNaseafter the RNA molecule was complexed with CNE particles. All sampleswere incubated with RNase for 30 minutes. RNase was inactivated withproteinase K and samples that were formulated were decomplexed andanalyzed for RNA integrity by denaturing gel electrophoresis. Unlabeledlanes contain molecular weight markers. Lanes 1 and 2: mouse thymus RNAbefore (1) and after (2) RNase digestion; lanes 3 and 4: mouse thymusRNA complexed with CNE35 at an N/P ratio of 10:1 before (3) and after(4) RNase digestion; lanes 5 and 6: mouse thymus RNA complexed withCNE35 at an N/P ratio of 4:1 before (5) and after (6) RNase digestion;lane 7: mouse thymus RNA before RNase digestion.

FIG. 7 shows the sequence of the vectors used in the examples. FIG. 7Ashows the sequence of plasmid A317 (SEQ ID NO:1), which encodes theRSV-F antigen.

FIG. 7B shows the sequence of plasmid A306 (SEQ ID NO:2), which encodessecreted human placental alkaline phosphatase (SEAP). FIG. 7C shows thesequence of plasmid A375 (SEQ ID NO:3), which encodes an RSV-F antigen.

FIG. 8A shows the results of the in vivo SEAP assay, using 1 μg of RNAreplicon A306 complexed with CNE17 at 10:1 N/P ratio. FIG. 8B the totalIgG titers in BALB/c mice at 2wp1 and 2wp2 time points (RNA repliconA317 complexed with CNE17 were administered to the BALB/c).

FIGS. 9A-9C show the effects of different buffer compositions onparticle size. FIG. 9A shows the effects of sugar, salt, and polymer F127 on particle size of CNE17 emulsions with RNA complexed at N/P of10:1. FIG. 9B shows the effect of citrate buffer on particle size ofCNE17 emulsion. FIG. 9C shows the effect of polymers (F68, F127 andPEG300) on particle size.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

This invention generally relates to cationic oil-in-water emulsions thatcan be used to deliver negatively charged molecules, such as an RNAmolecule to cells. The emulsion particles comprise an oil core and acationic lipid. The cationic lipid can interact with the negativelycharged molecule, for example through electrostatic forces andhydrophobic/hydrophilic interactions, thereby anchoring the molecule tothe emulsion particles. The cationic emulsions described herein areparticularly suitable for delivering nucleic acid molecules, such as anRNA molecules (e.g., RNA that encoding a protein or peptide, smallinterfering RNA, self-replicating RNA, and the like) to cells in vivo.

The present invention is based on the discovery that cationicoil-in-water emulsions can be used to deliver negatively chargedmolecules to cells. The emulsion particles comprise an oil core, and acationic lipid that can interact with the negatively charged molecule.In preferred embodiments, an RNA molecule is complexed, for examplethrough electrostatic forces and hydrophobic/hydrophilic interactions,with a particle of a cationic oil-in-water emulsion. The complexed RNAmolecule is stabilized and protected from RNase-mediated degradation,and is more efficiently taken up by cells relative to free RNA. Inaddition, when the RNA is delivered to induce expression of an encodedprotein, such as in the context of an RNA vaccine, the immunogenicity ofthe encoded protein can be enhanced due to adjuvant effects of theemulsion. Therefore, in addition to more efficient delivery of anegatively charged molecule (e.g., an RNA molecule that encodes anantigen), the cationic emulsions can also enhance the immune responsethrough adjuvant activity.

For example, as described and exemplified herein, the inventorsevaluated the in vivo effects of a series of cationic oil-in-wateremulsions, using a mouse model and a cotton rat model of respiratorysyncytial virus (RSV) immunization. The results demonstrate thatformulations in which the RNA molecules were complexed with cationicemulsions generated significantly higher immune responses as compared tofree RNA formulations. In some cases, the average antibody titersagainst an RNA encoded protein that were obtained followingadministration of 1 μg of RNA complexed with a cationic oil-in-wateremulsions, were comparable to titers obtained using 10 times more freeRNA (10 μg dose of free RNA). Another advantage of the formulations asdescribed herein, in addition to higher immune responses in the host, isthat there was less fluctuation in the immune responses in the hostanimals between different studies and different host animals, ascompared to free (unformulated) RNA.

Accordingly, in one aspect, the invention provides a compositioncomprising an RNA molecule complexed with a particle of a cationicoil-in-water emulsion, wherein the particle comprises (a) an oil corethat is in liquid phase at 25° C., and (b) a cationic lipid. Preferably,the cationic oil-in-water emulsion particle is not a Nano Lipid PeptideParticle (NLPP). Preferably, the oil core is in liquid phase at 4° C.

The cationic emulsion particles may further comprises a surfactant(e.g., Tween 80 (polysorbate 80; polyoxyethylenesorbitan monooleate),SPAN85 (sorbtian trioleate), or a combination thereof), a phospholipid,or a combination thereof. The emulsion may also comprise a polymer(e.g., Pluronic® F127) in the aqueous phase (the continuous phase) ofthe emulsion.

In another aspect, the invention also provides several specificformulations of cationic oil-in-water emulsions that can be used todeliver negatively charged molecules.

In another aspect, the invention provides a method of preparing acomposition that comprises a negatively charged molecule complexed witha particle of a cationic oil-in-water emulsion. One exemplary approachto produce cationic emulsions described herein is by dispersing the oilphase in the aqueous phase by homogenization. Additional optional stepsto promote particle formation, to improve the complexation between thenegatively charged molecules and the cationic particles, to increase thestability of the negatively charged molecule (e.g., to preventdegradation of an RNA molecule), to facilitate appropriatedecomplexation/release of the negatively charged molecules (such as anRNA molecule), or to prevent aggregation of the emulsion particlesinclude, for example, adding dichloromethane (DCM or methylene chloride)into the oil phase, and allowing DCM to evaporate before or afterhomogenization; mixing the cationic lipid with a suitable solvent toform a liposome suspension; or adding a polymer (e.g., Pluronic® F127)or a surfactant to the aqueous phase of the emulsion. Alternatively, thecationic lipid may be dissolved directly in the oil.

The cationic emulsions of the invention can be used to deliver anegatively charged molecule, such as a nucleic acid (e.g., RNA). Thecompositions may be administered to a subject in need thereof togenerate or potentiate an immune response. The compositions can also beco-delivered with another immunogenic molecule, immunogenic compositionor vaccine to enhance the effectiveness of the induced immune response.

2. Definitions

As used herein, the singular forms “a,” “an” and “the” include pluralreferences unless the content clearly dictates otherwise.

The term “about”, as used here, refers to +/−10% of a value.

The term “surfactant” is a term of art and generally refers to anymolecule having both a hydrophilic group (e.g., a polar group), whichenergetically prefers solvation by water, and a hydrophobic group whichis not well solvated by water. The term “nonionic surfactant” is a knownterm in the art and generally refers to a surfactant molecule whosehydrophilic group (e.g., polar group) is not electrostatically charged.

The term “polymer” refers to a molecule consisting of individualchemical moieties, which may be the same or different, that are joinedtogether. As used herein, the term “polymer” refers to individualchemical moieties that are joined end-to-end to form a linear molecule,as well as individual chemical moieties joined together in the form of abranched (e.g., a “multi-arm” or “star-shaped”) structure. Exemplarypolymers include, e.g., poloxamers. Poloxamers are nonionic triblockcopolymers having a central hydrophobic chain of polyoxypropylene(poly(propylene oxide)) flanked by two hydrophilic chains ofpolyoxyethylene (poly(ethylene oxide)).

A “buffer” refers to an aqueous solution that resists changes in the pHof the solution.

As used herein, “nucleotide analog” or “modified nucleotide” refers to anucleotide that contains one or more chemical modifications (e.g.,substitutions) in or on the nitrogenous base of the nucleoside (e.g.,cytosine (C), thymine (T) or uracil (U)), adenine (A) or guanine (G)). Anucleotide analog can contain further chemical modifications in or onthe sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modifiedribose, modified deoxyribose, six-membered sugar analog, or open-chainsugar analog), or the phosphate.

As use herein, “saccharide” encompasses monosaccharides,oligosaccharides, or polysaccharides in straight chain or ring forms, ora combination thereof to form a saccharide chain. Oligosaccharides aresaccharides having two or more monosaccharide residues. Examples ofsaccharides include glucose, maltose, maltotriose, maltotetraose,sucrose and trehalose.

The terms “self-replicating RNA,” “RNA replicon” or “RNA vector” is aterm of art and generally refer to an RNA molecule which is capable ofdirecting its own amplification or self-replication in vivo, typicallywithin a target cell. The RNA replicon is used directly, without therequirement for introduction of DNA into a cell and transport to thenucleus where transcription would occur. By using the RNA vector fordirect delivery into the cytoplasm of the host cell, autonomousreplication and translation of the heterologous nucleic acid sequenceoccurs efficiently. An alphavirus-derived self-replicating RNA maycontain the following elements in sequential order: 5′ viral sequencesrequired in cis for replication (also referred to as 5′ CSE, inbackground), sequences which, when expressed, code for biologicallyactive alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4),3′ viral sequences required in cis for replication (also referred to as3′ CSE, in background), and a polyadenylate tract. Thealphavirus-derived self-replicating RNA may also contain a viralsubgenomic “junction region” promoter, sequences from one or morestructural protein genes or portions thereof, extraneous nucleic acidmolecule(s) which are of a size sufficient to allow production ofrecombinant alphavirus particles, as well as heterologous sequence(s) tobe expressed.

The term “adjuvant” refers to any substance that assists or modifies theaction of a pharmaceutical, including but not limited to immunologicaladjuvants, which increase and/or diversify the immune response to anantigen. Hence, immunological adjuvants include compounds that arecapable of potentiating an immune response to antigens. Immunologicaladjuvants can potentiate humoral and/or cellular immunity. Substancesthat stimulate an innate immune response are included within thedefinition of immunological adjuvants herein Immunological adjuvants mayalso be referred to as “immunopotentiators.”

As used herein, an “antigen” or “immunogen” refers to a moleculecontaining one or more epitopes (e.g., linear, conformational or both)that elicit an immunological response. As used herein, an “epitope” isthat portion of given species (e.g., an antigenic molecule or antigeniccomplex) that determines its immunological specificity. An epitope iswithin the scope of the present definition of antigen. The term“antigen” or “immunogen” as used herein includes subunit antigens, i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature. Antibodies such asanti-idiotype antibodies, or fragments thereof, and synthetic peptidemimotopes, which can mimic an antigen or antigenic determinant, are alsocaptured under the definition of antigen as used herein.

An “immunological response” or “immune response” is the development in asubject of a humoral and/or a cellular immune response to an antigen oran immunological adjuvant.

Immune responses include innate and adaptive immune responses. Innateimmune responses are fast-acting responses that provide a first line ofdefense for the immune system. In contrast, adaptive immunity usesselection and clonal expansion of immune cells having somaticallyrearranged receptor genes (e.g., T- and B-cell receptors) that recognizeantigens from a given pathogen or disorder (e.g., a tumor), therebyproviding specificity and immunological memory. Innate immune responses,among their many effects, lead to a rapid burst of inflammatorycytokines and activation of antigen-presenting cells (APCs) such asmacrophages and dendritic cells. To distinguish pathogens fromself-components, the innate immune system uses a variety of relativelyinvariable receptors that detect signatures from pathogens, known aspathogen-associated molecular patterns, or PAMPs. The addition ofmicrobial components to experimental vaccines is known to lead to thedevelopment of robust and durable adaptive immune responses. Themechanism behind this potentiation of the immune responses has beenreported to involve pattern-recognition receptors (PRRs), which aredifferentially expressed on a variety of immune cells, includingneutrophils, macrophages, dendritic cells, natural killer cells, B cellsand some nonimmune cells such as epithelial and endothelial cells.Engagement of PRRs leads to the activation of some of these cells andtheir secretion of cytokines and chemokines, as well as maturation andmigration of other cells. In tandem, this creates an inflammatoryenvironment that leads to the establishment of the adaptive immuneresponse. PRRs include nonphagocytic receptors, such as Toll-likereceptors (TLRs) and nucleotide-binding oligomerization domain (NOD)proteins, and receptors that induce phagocytosis, such as scavengerreceptors, mannose receptors and (3-glucan receptors. Reported TLRs(along with examples of some reported ligands, which may be used asimmunogenic molecule in various embodiments of the invention) includethe following: TLR1 (bacterial lipoproteins from Mycobacteria,Neisseria), TLR2 (zymosan yeast particles, peptidoglycan, lipoproteins,lipopeptides, glycolipids, lipopolysaccharide), TLR3 (viraldouble-stranded RNA, poly:IC), TLR4 (bacterial lipopolysaccharides,plant product taxol), TLR5 (bacterial flagellins), TLR6 (yeast zymosanparticles, lipotechoic acid, lipopeptides from mycoplasma), TLR7(single-stranded RNA, imiquimod, resimiquimod, and other syntheticcompounds such as loxoribine and bropirimine), TLR8 (single-strandedRNA, resimiquimod) and TLR9 (CpG oligonucleotides), among others.Dendritic cells are recognized as some of the most important cell typesfor initiating the priming of naive CD4⁺ helper T (T_(H)) cells and forinducing CD8⁺ T cell differentiation into killer cells. TLR signalinghas been reported to play an important role in determining the qualityof these helper T cell responses, for instance, with the nature of theTLR signal determining the specific type of T_(H) response that isobserved (e.g., T_(H)1 versus T_(H)2 response). A combination ofantibody (humoral) and cellular immunity are produced as part of aT_(H1)-type response, whereas a T_(H)2-type response is predominantly anantibody response. Various TLR ligands such as CpG DNA (TLR9) andimidazoquinolines (TLR7, TLR8) have been documented to stimulatecytokine production from immune cells in vitro. The imidazoquinolinesare the first small, drug-like compounds shown to be TLR agonists. Forfurther information, see, e.g., A. Pashine, N. M. Valiante and J. B.Ulmer, Nature Medicine 11, S63-S68 (2005), K. S. Rosenthal and D. HZimmerman, Clinical and Vaccine Immunology, 13(8), 821-829 (2006), andthe references cited therein.

For purposes of the present invention, a humoral immune response refersto an immune response mediated by antibody molecules, while a cellularimmune response is one mediated by T-lymphocytes and/or other whiteblood cells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (CTLs). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote theintracellular destruction of intracellular microbes, or the lysis ofcells infected with such microbes. Another aspect of cellular immunityinvolves an antigen-specific response by helper T-cells. Helper T-cellsact to help stimulate the function, and focus the activity of,nonspecific effector cells against cells displaying peptide antigens inassociation with MHC molecules on their surface. A “cellular immuneresponse” also refers to the production of cytokines, chemokines andother such molecules produced by activated T-cells and/or other whiteblood cells, including those derived from CD4⁺ and CD8⁺ T-cells.

A composition such as an immunogenic composition or a vaccine thatelicits a cellular immune response may thus serve to sensitize avertebrate subject by the presentation of antigen in association withMHC molecules at the cell surface. The cell-mediated immune response isdirected at, or near, cells presenting antigen at their surface. Inaddition, antigen-specific T-lymphocytes can be generated to allow forthe future protection of an immunized host. The ability of a particularantigen or composition to stimulate a cell-mediated immunologicalresponse may be determined by a number of assays known in the art, suchas by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, by assaying for T-lymphocytes specific for the antigen in asensitized subject, or by measurement of cytokine production by T cellsin response to restimulation with antigen. Such assays are well known inthe art. See, e.g., Erickson et al. (1993) J. Immunol. 151:4189-4199;Doe et al. (1994) Eur. J. Immunol. 24:2369-2376. Thus, an immunologicalresponse as used herein may be one which stimulates the production ofCTLs and/or the production or activation of helper T-cells. The antigenof interest may also elicit an antibody-mediated immune response. Hence,an immunological response may include, for example, one or more of thefollowing effects among others: the production of antibodies by, forexample, B-cells; and/or the activation of suppressor T-cells and/or yδT-cells directed specifically to an antigen or antigens present in thecomposition or vaccine of interest. These responses may serve, forexample, to neutralize infectivity, and/or mediate antibody-complement,or antibody dependent cell cytotoxicity (ADCC) to provide protection toan immunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

Compositions in accordance with the present invention display “enhancedimmunogenicity” for a given antigen when they possess a greater capacityto elicit an immune response than the immune response elicited by anequivalent amount of the antigen in a differing composition (e.g.,wherein the antigen is administered as a soluble protein). Thus, acomposition may display enhanced immunogenicity, for example, becausethe composition generates a stronger immune response, or because a lowerdose or fewer doses of antigen is necessary to achieve an immuneresponse in the subject to which it is administered. Such enhancedimmunogenicity can be determined, for example, by administering acomposition of the invention and an antigen control to animals andcomparing assay results of the two.

3. Cationic Oil-in-Water Emulsions

The cationic oil-in-water emulsions disclosed herein are generallydescribed in the manner that is conventional in the art, byconcentrations of components that are used to prepare the emulsions. Itis understood in the art that during the process of producing emulsions,including sterilization and other downstream processes, small amounts ofoil (e.g., squalene), cationic lipid (e.g., DOTAP), or other componentsmay be lost, and the actual concentrations of these components in thefinal product (e.g., a packaged, sterilized emulsion that is ready foradministration) might be slightly lower than starting amounts, sometimesby up to about 10% or by up to about 20%.

This invention generally relates to cationic oil-in-water emulsions thatcan be used to deliver negatively charged molecules, such as an RNAmolecule. The emulsion particles comprise an oil core and a cationiclipid. The cationic lipid can interact with the negatively chargedmolecule, for example through electrostatic forces andhydrophobic/hydrophilic interactions, thereby anchoring the molecule tothe emulsion particles. The cationic emulsions described herein areparticularly suitable for delivering a negatively charged molecule, suchas an RNA molecule encoding an antigen or small interfering RNA to cellsin vivo. For example, the cationic emulsions described herein provideadvantages for delivering RNA that encode antigens, includingself-replicating RNAs, as vaccines.

The particles of the oil-in-water emulsions resemble a micelle with acentral core of oil. The oil core is coated with the cationic lipid,which disperses the oil droplet in the aqueous (continuous) phase asmicelle-like droplets. One or more optional components may be present inthe emulsion, such as surfactants and/or phospholipids as describedbelow. For example, one or more surfactants may be used to promoteparticle formation and/or to stabilize the emulsion particles. In thatcase, the oil core is coated with the cationic lipid as well as thesurfactant(s) to form micelle-like droplets. Similarly, one or morelipids (e.g., neutral lipids, glycol-lipids or phospholipids) may alsobe present on the surface of the emulsion particles, if such lipids areused as emulsifiers to disperse the oil droplets.

The particles of the oil-in-water emulsions have an average diameter(i.e., the number average diameter) of 1 micrometer or less. It isparticularly desirable that the average particle size (i.e., the numberaverage diameter) of the cationic emulsions is about 900 nm or less,about 800 nm or less, about 700 nm or less, about 600 nm or less, about500 nm or less, about 400 nm or less, 300 nm or less, or 200 nm or less,for example, from about 1 nm to about 1 μm, from about 1 nm to about 900nm, from about 1 nm to about 800 nm, from about 1 nm to about 700 nm,from about 1 nm to about 600 nm, from about 1 nm to about 500 nm, fromabout 1 nm to about 400 nm, from about 1 nm to about 300 nm, from about1 nm to about 200 nm, from about 1 nm to about 175 nm, from about 1 nmto about 150 nm, from about 1 nm to about 125 nm, from about 1 nm toabout 100 nm, from about 1 nm to about 75 nm, or from about 1 nm toabout 50 nm.

It is particularly desirable that the average particle diameter of thecationic emulsions is about 180 nm or less, about 170 nm or less, about160 nm or less, about 150 nm or less, about 140 nm or less, about 130 nmor less, about 120 nm or less, about 110 nm or less, or about 100 nm orless; for example, from about 80 nm to 180 nm, from about 80 nm to 170nm, from about 80 nm to 160 nm, from about 80 nm to 150 nm, from about80 nm to 140 nm, from about 80 nm to 130 nm, from about 80 nm to 120 nm;from about 80 nm to 110 nm, or from about 80 nm to 100 nm. Particularlypreferred average particle diameter is about 100 nm.

The size of the emulsion particles can be varied by changing the ratioof surfactant to oil (increasing the ratio decreases droplet size),operating pressure of homogenization (increasing operating pressure ofhomogenization typically reduces droplet size), temperature (increasingtemperature decreases droplet size), changing the type of oil, and otherprocess parameters, as described in detail below. Inclusion of certaintypes of buffers in the aqueous phase may also affect the particle size.

In some cases, in the context of an RNA vaccine, the size of theemulsion particles may affect the immunogenicity of the RNA-emulsioncomplex. Therefore, the preferred range of the average particle size foremulsions should be from about 80 nm to about 180 nm in diameter.

The emulsion particles described herein can be complexed with anegatively charged molecule. Prior to complexation with the negativelycharged molecule, the overall net charge of the particles (typicallymeasured as zeta-potential) should be positive (cationic). The overallnet charge of the particles may vary, depending on the type of thecationic lipid and the amount of the cationic lipid in the emulsion, theamount of oil in the emulsion (e.g. higher percentage of oil typicallyresults in less charge on the surface of the particles), and may also beaffected by any additional component (e.g., surfactant(s) and/orphospholipid(s)) that is present in the emulsion. In the exemplaryembodiments, the zeta-potential of the pre-complexation particles aretypically above 10 mV.

Preferably, the zeta-potential of the pre-complexation particles are nomore than about 50 mV, no more than about 45 mV, no more than about 40mV, no more than about 35 mV, no more than about 30 mV, no more thanabout 25 mV, no more than about 20 mV; from about 5 mV to about 50 mV,from about 10 mV to about 50 mV, from about 10 mV to about 45 mV, fromabout 10 mV to about 40 mV, from about 10 mV to about 35 mV, from about10 mV to about 30 mV, from about 10 mV to about 25 mV, or from about 10mV to about 20 mV. Zeta potential can be affected by (i) pH of theemulsion, (ii) conductivity of the emulsion (e.g., salinity), and (iii)the concentration of the various components of the emulsion (polymer,non-ionic surfactants etc.). Zeta potential of CNEs is measured using aMalvern Nanoseries Zetasizer (Westborough, Mass.). The sample is diluted1:100 in water (viscosity: 0.8872 cp, RI: 1.330, Dielectric constant:78.5) and is added to a polystyrene latex capillary cell (Malvern,Westborough, Mass.). Zeta potential is measured at 25° C. with a 2minute equilibration time and analyzed using the Smoluchowski model(F(Ka) value=1.5). Data is reported in mV.

An exemplary cationic emulsion of the invention is CNE17. The oil coreof CNE17 is squalene (at 4.3% w/v) and the cationic lipid is DOTAP (at1.4 mg/mL). CNE17 also includes the surfactants SPAN85 ((sorbtiantrioleate) at 0.5% v/v) and Tween 80 ((polysorbate 80;polyoxyethylenesorbitan monooleate) at 0.5% v/v). Thus, the particles ofCNE17 comprise a squalene core coated with SPAN85, Tween80, and DOTAP.RNA molecules were shown to complex with CNE17 particles efficiently at4:1 N/P ratio and 10:1 N/P ratio. Other exemplary cationic emulsionsinclude, e.g., CNE05 (0.5% w/v squalene, 0.08% Tween 80, and 1.2 mg/mLDOTAP), CNE12 (4.3% squalene, 0.5% SPAN85, 0.5% Tween 80, and 2.46 mg/mLDC Cholesterol), CNE13 (4.3% squalene, 0.5% SPAN85, 0.5% Tween 80, and1.45 mg/mL DDA), and other emulsions described herein.

The individual components of the oil-in-water emulsions of the presentinvention are known in the art, although such compositions have not beencombined in the manner described herein. Accordingly, the individualcomponents, although described below both generally and in some-detailfor preferred embodiments, are well known in the art, and the terms usedherein, such as oil core, surfactant, phospholipids, etc., aresufficiently well known to one skilled in the art without furtherdescription. In addition, while preferred ranges of the amount of theindividual components of the emulsions are provided, the actual ratiosof the components of a particular emulsion may need to be adjusted suchthat emulsion particles of desired size and physical property can beproperly formed. For example, if a particular amount of oil is used(e.g. 5% v/v oil), then, the amount of surfactant should be at levelthat is sufficient to disperse the oil droplet into aqueous phase toform a stable emulsion. The actual amount of surfactant required todisperse the oil droplet into aqueous phase depends on the type ofsurfactant and the type of oil core used for the emulsion; and theamount of oil may also vary according to droplet size (as this changesthe surface area between the two phases). The actual amounts and therelative proportions of the components of a desired emulsion can bereadily determined by a skilled artisan.

A. Oil Core

The particles of the cationic oil-in-water emulsions comprise an oilcore. Preferably, the oil is a metabolizable, non-toxic oil; morepreferably one of about 6 to about 30 carbon atoms including, but notlimited to, alkanes, alkenes, alkynes, and their corresponding acids andalcohols, the ethers and esters thereof, and mixtures thereof. The oilmay be any vegetable oil, fish oil, animal oil or synthetically preparedoil that can be metabolized by the body of the subject to which theemulsion will be administered, and is not toxic to the subject. Thesubject may be an animal, typically a mammal, and preferably a human.

In certain embodiments, the oil core is in liquid phase at 25° C. Theoil core is in liquid phase at 25° C., when it displays the propertiesof a fluid (as distinguished from solid and gas; and having a definitevolume but no definite shape) when stored at 25° C. The emulsion,however, may be stored and used at any suitable temperature. Preferably,the oil core is in liquid phase at 4° C.

The oil may be any long chain alkane, alkene or alkyne, or an acid oralcohol derivative thereof either as the free acid, its salt or an estersuch as a mono-, or di- or triester, such as the triglycerides andesters of 1,2-propanediol or similar poly-hydroxy alcohols. Alcohols maybe acylated employing a mono- or poly-functional acid, for exampleacetic acid, propanoic acid, citric acid or the like. Ethers derivedfrom long chain alcohols which are oils and meet the other criteria setforth herein may also be used.

The individual alkane, alkene or alkyne moiety and its acid or alcoholderivatives will generally have from about 6 to about 30 carbon atoms.The moiety may have a straight or branched chain structure. It may befully saturated or have one or more double or triple bonds. Where monoor poly ester- or ether-based oils are employed, the limitation of about6 to about 30 carbons applies to the individual fatty acid or fattyalcohol moieties, not the total carbon count.

It is particularly desirable that the oil can be metabolized by the hostto which the emulsion is administered.

Any suitable oils from an animal, fish or vegetable source may be used.Sources for vegetable oils include nuts, seeds and grains, and suitableoils peanut oil, soybean oil, coconut oil, and olive oil and the like.Other suitable seed oils include safflower oil, cottonseed oil,sunflower seed oil, sesame seed oil and the like. In the grain group,corn oil, and the oil of other cereal grains such as wheat, oats, rye,rice, teff, triticale and the like may also be used. The technology forobtaining vegetable oils is well developed and well known. Thecompositions of these and other similar oils may be found in, forexample, the Merck Index, and source materials on foods, nutrition andfood technology.

About six to about ten carbon fatty acid esters of glycerol and1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. These products arecommercially available under the name NEOBEES from PVO International,Inc., Chemical Specialties Division, 416 Division Street, Boongon, N.J.and others.

Animal oils and fats are often in solid phase at physiologicaltemperatures due to the fact that they exist as triglycerides and have ahigher degree of saturation than oils from fish or vegetables. However,fatty acids are obtainable from animal fats by partial or completetriglyceride saponification which provides the free fatty acids. Fatsand oils from mammalian milk are metabolizable and may therefore be usedin the practice of this invention. The procedures for separation,purification, saponification and other means necessary for obtainingpure oils from animal sources are well known in the art.

Most fish contain metabolizable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene),a branched, unsaturated terpenoid, is particularly preferred herein. Amajor source of squalene is shark liver oil, although plant oils(primarily vegetable oils), including amaranth seed, rice bran, wheatgerm, and olive oils, are also suitable sources. Squalane, the saturatedanalog to squalene, is also preferred. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art.

In certain embodiments, the oil core comprises an oil that is selectedfrom the group consisting of: Castor oil, Coconut oil, Corn oil,Cottonseed oil, Evening primrose oil, Fish oil, Jojoba oil, Lard oil,Linseed oil, Olive oil, Peanut oil, Safflower oil, Sesame oil, Soybeanoil, Squalene, Sunflower oil, Wheatgerm oil, and Mineral oil. Inexemplary embodiments, the oil core comprises Soybean oil, Sunfloweroil, Olive oil, Squalene, or a combination thereof. Squalane can also beused as the oil. In exemplary embodiments, the oil core comprisesSqualene, Squalane, or a combination thereof.

The oil component of the emulsion may be present in an amount from about0.2% to about 20% (v/v). For example, the cationic oil-in-water emulsionmay comprise from about 0.2% to about 20% (v/v) oil, from about 0.2% toabout 15% (v/v) oil, from about 0.2% to about 10% (v/v) oil, from about0.2% to about 9% (v/v) oil, from about 0.2% to about 8% (v/v) oil, fromabout 0.2% to about 7% (v/v) oil, from about 0.2% to about 6% (v/v) oil,from about 0.2% to about 5% (v/v) oil, from about 0.2% to about 4.3%(v/v) oil, from about 0.3% to about 20% (v/v) oil, from about 0.4% toabout 20% (v/v) oil, from about 0.5% to about 20% (v/v) oil, from about1% to about 20% (v/v) oil, from about 2% to about 20% (v/v) oil, fromabout 3% to about 20% (v/v) oil, from about 4% to about 20% (v/v) oil,from about 4.3% to about 20% (v/v) oil, from about 5% to about 20% (v/v)oil, about 0.5% (v/v) oil, about 1% (v/v) oil, about 1.5% (v/v) oil,about 2% (v/v) oil, about 2.5% (v/v) oil, about 3% (v/v) oil, about 3.5%(v/v) oil, about 4% (v/v) oil, about 4.3% (v/v) oil, about 5% (v/v) oil,or about 10% (v/v) oil.

Alternatively, the cationic oil-in-water emulsion may comprise fromabout 0.2% to about 10% (w/v) oil, from about 0.2% to about 9% (w/v)oil, from about 0.2% to about 8% (w/v) oil, from about 0.2% to about 7%(w/v) oil, from about 0.2% to about 6% (w/v) oil, from about 0.2% toabout 5% (w/v) oil, from about 0.2% to about 4.3% (w/v) oil, or about4.3% (w/v) oil.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesabout 0.5% (v/v) oil. In another exemplary embodiment, the cationicoil-in-water emulsion comprises about 4.3% (v/v) oil. In anotherexemplary embodiment, the cationic oil-in-water emulsion comprises about5% (v/v) oil. In another exemplary embodiment, the cationic oil-in-wateremulsion comprises about 4.3% (w/v) squalene.

As noted above, the percentage of oil described above is determinedbased on the initial amount of the oil that is used to prepare theemulsions. It is understood in the art that the actual concentration ofthe oil in the final product (e.g., a packaged, sterilized emulsion thatis ready for administration) might be slightly lower, sometimes up toabout 10% or about 20%.

B. Cationic Lipids

The emulsion particles described herein comprise a cationic lipid, whichcan interact with the negatively charged molecule thereby anchoring themolecule to the emulsion particles.

Any suitable cationic lipid may be used. Generally, the cationic lipidcontains a nitrogen atom that is positively charged under physiologicalconditions. Suitable cationic lipids include, benzalkonium chloride(BAK), benzethonium chloride, cetrimide (which containstetradecyltrimethylammonium bromide and possibly small amounts ofdodecyltrimethylammonium bromide and hexadecyltrimethyl ammoniumbromide), cetylpyridinium chloride (CPC), cetyl trimethylammoniumchloride (CTAC), primary amines, secondary amines, tertiary amines,including but not limited to N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, other quaternary amine salts,including but not limited to dodecyltrimethylammonium bromide,hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammoniumbromide, benzyldimethyldodecylammonium chloride,benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammoniummethoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethoniumchloride, methyl mixed trialkyl ammonium chloride, methyltrioctylammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3tetramethylbutyl)-phenoxy]-ethoxy)ethyl]-benzenemetha-naminiumchloride (DEBDA), dialkyldimethylammonium salts,[1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride,1,2-diacyl-3-(trimethylammonio) propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3(dimethylammonio)propane(acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl),1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl3-succinyl-sn-glycerol choline ester, cholesteryl (4′-trimethylammonio)butanoate), N-alkyl pyridinium salts (e.g. cetylpyridinium bromide andcetylpyridinium chloride), N-alkylpiperidinium salts, dicationicbolaform electrolytes (C₁₂Me₆; C₁₂Bu₆),dialkylglycetylphosphorylcholine, lysolecithin, L-αdioleoylphosphatidylethanolamine, cholesterol hemisuccinate cholineester, lipopolyamines, including but not limited todioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine(LPLL, LPDL), poly (L (or D)-lysine conjugated toN-glutarylphosphatidylethanolamine, didodecyl glutamate ester withpendant amino group (C₁₂GluPhC_(n)N⁺), ditetradecyl glutamate ester withpendant amino group (C₁₄GluC_(n)N⁺), cationic derivatives ofcholesterol, including but not limited tocholesteryl-3β-oxysuccinamidoethylenetrimethylammonium salt,cholesteryl-3β-oxysuccinamidoethylenedimethylamine,cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt,cholesteryl-3β-carboxyamidoethylenedimethylamine, and3γ-[N—(N′,N-dimethylaminoetanecarbomoyl]cholesterol) (DC-Cholesterol),1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), and combination thereof.

Other cationic lipids suitable for use in the invention include, e.g.,the cationic lipids described in U.S. Patent Publications 2008/0085870(published Apr. 10, 2008) and 2008/0057080 (published Mar. 6, 2008).

Other cationic lipids suitable for use in the invention include, e.g.,Lipids E0001-E0118 or E0119-E0180 as disclosed in Table 6 (pages112-139) of WO 2011/076807 (which also discloses methods of making, andmethod of using these cationic lipids). Additional suitable cationiclipids include N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA).

The emulsion may comprise any combination of two or more of the cationiclipids described herein.

In preferred embodiments, the cationic lipid is selected from the groupconsisting of 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),313-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), Lipids E0001-E0118 orE0119-E0180 as disclosed in Table 6 (pages 112-139) of WO 2011/076807,and combinations thereof.

In other preferred embodiments, the cationic lipid is selected from thegroup consisting of 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),Dimethylaminoethane)-carbamoyl]Cholesterol (DC Cholesterol),dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), Lipids E0001-E0118or E0119-E0180 as disclosed in Table 6 (pages 112-139) of WO2011/076807, and combinations thereof.

In certain embodiments, the cationic lipid is DOTAP. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 25mg/ml DOTAP. For example, the cationic oil-in-water emulsion maycomprise DOTAP at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, from about 0.5mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8 mg/ml, fromabout 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml to about 1.6mg/ml, from about 0.6 mg/ml to about 1.6 mg/ml, from about 0.7 mg/ml toabout 1.6 mg/ml, from about 0.8 mg/ml to about 1.6 mg/ml, from about 0.8mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5mg/ml, about 1.6 mg/ml, about 12 mg/ml, about 18 mg/ml, about 20 mg/ml,about 21.8 mg/ml, about 24 mg/ml, etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.6 mg/ml DOTAP, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In certain embodiments, the cationic lipid is DC Cholesterol. Thecationic oil-in-water emulsion may comprise DC Cholesterol at from about0.1 mg/ml to about 5 mg/ml DC Cholesterol. For example, the cationicoil-in-water emulsion may comprise DC Cholesterol from about 0.1 mg/mlto about 5 mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3mg/ml to about 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, fromabout 0.5 mg/ml to about 5 mg/ml, from about 0.62 mg/ml to about 5mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1.5 mg/ml toabout 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about 2.46mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, from about3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml, fromabout 4.5 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.92mg/ml, from about 0.1 mg/ml to about 4.5 mg/ml, from about 0.1 mg/ml toabout 4 mg/ml, from about 0.1 mg/ml to about 3.5 mg/ml, from about 0.1mg/ml to about 3 mg/ml, from about 0.1 mg/ml to about 2.46 mg/ml, fromabout 0.1 mg/ml to about 2 mg/ml, from about 0.1 mg/ml to about 1.5mg/ml, from about 0.1 mg/ml to about 1 mg/ml, from about 0.1 mg/ml toabout 0.62 mg/ml, about 0.15 mg/ml, about 0.3 mg/ml, about 0.6 mg/ml,about 0.62 mg/ml, about 0.9 mg/ml, about 1.2 mg/ml, about 2.46 mg/ml,about 4.92 mg/ml, etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.62 mg/ml to about 4.92 mg/ml DC Cholesterol, such as 2.46mg/ml.

In certain embodiments, the cationic lipid is DDA. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 5 mg/mlDDA. For example, the cationic oil-in-water emulsion may comprise DDA atfrom about 0.1 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.5mg/ml, from about 0.1 mg/ml to about 4 mg/ml, from about 0.1 mg/ml toabout 3.5 mg/ml, from about 0.1 mg/ml to about 3 mg/ml, from about 0.1mg/ml to about 2.5 mg/ml, from about 0.1 mg/ml to about 2 mg/ml, fromabout 0.1 mg/ml to about 1.5 mg/ml, from about 0.1 mg/ml to about 1.45mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3 mg/ml toabout 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, from about 0.5mg/ml to about 5 mg/ml, from about 0.6 mg/ml to about 5 mg/ml, fromabout 0.73 mg/ml to about 5 mg/ml, from about 0.8 mg/ml to about 5mg/ml, from about 0.9 mg/ml to about 5 mg/ml, from about 1.0 mg/ml toabout 5 mg/ml, from about 1.2 mg/ml to about 5 mg/ml, from about 1.45mg/ml to about 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about2.5 mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, fromabout 3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml,from about 4.5 mg/ml to about 5 mg/ml, about 1.2 mg/ml, about 1.45mg/ml, etc. Alternatively, the cationic oil-in-water emulsion maycomprise DDA at about 20 mg/ml, about 21 mg/ml, about 21.5 mg/ml, about21.6 mg/ml, about 25 mg/ml.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.73 mg/ml to about 1.45 mg/ml DDA, such as 1.45 mg/ml.

In certain embodiments, the cationic lipid is DOTMA. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 25mg/ml DOTMA. For example, the cationic oil-in-water emulsion maycomprise DOTMA at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, from about 0.5mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8 mg/ml, fromabout 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml to about 1.6mg/ml, from about 0.6 mg/ml to about 1.6 mg/ml, from about 0.7 mg/ml toabout 1.6 mg/ml, from about 0.8 mg/ml to about 1.6 mg/ml, from about 0.8mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.35 mg/ml, about 1.4mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 12 mg/ml, about 18 mg/ml,about 20 mg/ml, about 22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.6 mg/ml DOTMA, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In certain embodiments, the cationic lipid is DOEPC. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 25mg/ml DOEPC. For example, the cationic oil-in-water emulsion maycomprise DOEPC at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 4 mg/ml, from about 0.5mg/ml to about 3 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, fromabout 0.5 mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8mg/ml, from about 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml toabout 1.6 mg/ml, from about 0.6 mg/ml to about 1.7 mg/ml, from about 0.7mg/ml to about 1.7 mg/ml, from about 0.8 mg/ml to about 1.7 mg/ml, fromabout 0.8 mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml,about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml,about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml,about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml,about 1.9 mg/ml, about 2.0 mg/ml, about 12 mg/ml, about 18 mg/ml, about20 mg/ml, about 22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.8 mg/ml DOEPC, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml, 1.6 mg/ml, 1.7 mg/ml, or 1.8 mg/ml.

In certain embodiments, the cationic lipid is DSTAP. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 50mg/ml DSTAP. For example, the cationic oil-in-water emulsion maycomprise DSTAP at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 4 mg/ml, from about 0.5mg/ml to about 3 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, fromabout 0.5 mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8mg/ml, from about 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml toabout 1.6 mg/ml, from about 0.6 mg/ml to about 1.7 mg/ml, from about 0.7mg/ml to about 1.7 mg/ml, from about 0.8 mg/ml to about 1.7 mg/ml, fromabout 0.8 mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml,about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml,about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml,about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml,about 1.9 mg/ml, about 2.0 mg/ml, about 12 mg/ml, about 18 mg/ml, about20 mg/ml, about 22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.6 mg/ml DSTAP, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In certain embodiments, the cationic lipid is DODAC. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 50mg/ml DODAC. For example, the cationic oil-in-water emulsion maycomprise DODAC at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 4 mg/ml, from about 0.5mg/ml to about 3 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, fromabout 0.5 mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8mg/ml, from about 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml toabout 1.6 mg/ml, from about 0.6 mg/ml to about 1.7 mg/ml, from about 0.7mg/ml to about 1.7 mg/ml, from about 0.8 mg/ml to about 1.7 mg/ml, fromabout 0.8 mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml,about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml,about 1.1 mg/ml, about 1.15 mg/ml, about 1.16 mg/ml, about 1.17 mg/ml,about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml,about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg/ml,about 2.0 mg/ml, about 12 mg/ml, about 18 mg/ml, about 20 mg/ml, about22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom 0.73 mg/ml to about 1.45 mg/ml DODAC, such as 1.45 mg/ml.

In certain embodiments, the cationic lipid is DODAP. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 50mg/ml DODAP. For example, the cationic oil-in-water emulsion maycomprise DODAP at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 4 mg/ml, from about 0.5mg/ml to about 3 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, fromabout 0.5 mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8mg/ml, from about 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml toabout 1.6 mg/ml, from about 0.6 mg/ml to about 1.7 mg/ml, from about 0.7mg/ml to about 1.7 mg/ml, from about 0.8 mg/ml to about 1.7 mg/ml, fromabout 0.8 mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml,about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml,about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml,about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml,about 1.9 mg/ml, about 2.0 mg/ml, about 12 mg/ml, about 18 mg/ml, about20 mg/ml, about 22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.6 mg/ml DODAP, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In some cases, it may be desirable to use a cationic lipid that issoluble in the oil core. For example, DOTAP DOEPC, DODAC, and DOTMA aresoluble in squalene or squalane. In other cases, it may be desirable touse a cationic lipid that is not soluble in the oil core. For example,DDA and DSTAP is not soluble in squalene. It is within the knowledge inthe art to determine whether a particular lipid is soluble or insolublein the oil and choose an appropriate oil and lipid combinationaccordingly. For example, solubility can be predicted based on thestructures of the lipid and oil (e.g., the solubility of a lipid may bedetermined by the structure of its tail). For example, lipids having oneor two unsaturated fatty acid chains (e.g., oleoyl tails), such asDOTAP, DOEPC, DODAC, DOTMA, are soluble in squalene or squalane; whereaslipids having saturated fatty acid chains (e.g., stearoyl tails) are notsoluble in squalene. Alternatively, solubility can be determinedaccording to the quantity of the lipid that dissolves in a givenquantity of the oil to form a saturated solution).

As noted above, the concentration of a lipid described above isdetermined based on the initial amount of the lipid that is used toprepare the emulsions. It is understood in the art that the actualconcentration of the oil in the final product (e.g., a packaged,sterilized emulsion that is ready for administration) might be slightlylower, sometimes by up to about 20%.

C. Additional Components

The cationic oil-in-water emulsions described herein may furthercomprise additional components. For example, the emulsions may comprisecomponents that can promote particle formation, improve the complexationbetween the negatively charged molecules and the cationic particles, orincrease the stability of the negatively charged molecule (e.g., toprevent degradation of an RNA molecule).

Surfactants

In certain embodiments, the particles of the cationic oil-in-wateremulsion further comprise a surfactant.

A substantial number of surfactants have been used in the pharmaceuticalsciences. These include naturally derived materials such as gums fromtrees, vegetable protein, sugar-based polymers such as alginates andcellulose, and the like. Certain oxypolymers or polymers having ahydroxide or other hydrophilic substituent on the carbon backbone havesurfactant activity, for example, povidone, polyvinyl alcohol, andglycol ether-based mono- and poly-functional compounds. Long chainfatty-acid-derived compounds form a third substantial group ofemulsifying and suspending agents which could be used in this invention.

Specific examples of suitable surfactants include the following:

1. Water-soluble soaps, such as the sodium, potassium, ammonium andalkanol-ammonium salts of higher fatty acids (C₁₀-C₂₂), in particularsodium and potassium tallow and coconut soaps.

2. Anionic synthetic non-soap surfactants, which can be represented bythe water-soluble salts of organic sulfuric acid reaction productshaving in their molecular structure an alkyl radical containing fromabout 8 to 22 carbon atoms and a radical selected from the groupconsisting of sulfonic acid and sulfuric acid ester radicals. Examplesof these are the sodium or potassium alkyl sulfates, derived from tallowor coconut oil; sodium or potassium alkyl benzene sulfonates; sodiumalkyl glyceryl ether sulfonates; sodium coconut oil fatty acidmonoglyceride sulfonates and sulfates; sodium or potassium salts ofsulfuric acid esters of the reaction product of one mole of a higherfatty alcohol and about 1 to 6 moles of ethylene oxide; sodium orpotassium alkyl phenol ethylene oxide ether sulfonates, with 1 to 10units of ethylene oxide per molecule and in which the alkyl radicalscontain from 8 to 12 carbon atoms; the reaction product of fatty acidsesterified with isethionic acid and neutralized with sodium hydroxide;sodium or potassium salts of fatty acid amide of a methyl tauride; andsodium and potassium salts of SO₃-sulfonated C₁₀-C₂₄ α-olefins.

3. Nonionic synthetic surfactants made by the condensation of alkyleneoxide groups with an organic hydrophobic compound. Typical hydrophobicgroups include condensation products of propylene oxide with propyleneglycol, alkyl phenols, condensation product of propylene oxide andethylene diamine, aliphatic alcohols having 8 to 22 carbon atoms, andamides of fatty acids.

4. Nonionic surfactants, such as amine oxides, phosphine oxides andsulfoxides, having semipolar characteristics. Specific examples of longchain tertiary amine oxides include dimethyldodecylamine oxide andbis-(2-hydroxyethyl) dodecylamine Specific examples of phosphine oxidesare found in U.S. Pat. No. 3,304,263, issued Feb. 14, 1967, and includedimethyldodecylphosphine oxide and dimethyl-(2hydroxydodecyl) phosphineoxide.

5. Long chain sulfoxides, including those corresponding to the formulaR¹—SO—R² wherein R¹ and R² are substituted or unsubstituted alkylradicals, the former containing from about 10 to about 28 carbon atoms,whereas R² contains from 1 to 3 carbon atoms. Specific examples of thesesulfoxides include dodecyl methyl sulfoxide and 3-hydroxy tridecylmethyl sulfoxide.

6. Ampholytic synthetic surfactants, such as sodium3-dodecylaminopropionate and sodium 3-dodecylaminopropane sulfonate.

7. Zwitterionic synthetic surfactants, such as3-(N,N-dimethyl-N-hexadecylammonio)propane-1-sulfonate and3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxy propane-1-sulfonate.

Additionally, all of the following types of surfactants can be used in acomposition of the present invention: (a) soaps (i.e., alkali salts) offatty acids, rosin acids, and tall oil; (b) alkyl arene sulfonates; (c)alkyl sulfates, including surfactants with both branched-chain andstraight-chain hydrophobic groups, as well as primary and secondarysulfate groups; (d) sulfates and sulfonates containing an intermediatelinkage between the hydrophobic and hydrophilic groups, such as thefatty acylated methyl taurides and the sulfated fatty monoglycerides;(e) long-chain acid esters of polyethylene glycol, especially the talloil esters; (f) polyethylene glycol ethers of alkylphenols; (g)polyethylene glycol ethers of long-chain alcohols and mercaptans; and(h) fatty acyl diethanol amides. Since surfactants can be classified inmore than one manner, a number of classes of surfactants set forth inthis paragraph overlap with previously described surfactant classes.

There are a number of surfactants specifically designed for and commonlyused in biological situations. Such surfactants are divided into fourbasic types: anionic, cationic, zwitterionic (amphoteric), and nonionic.Exemplary anionic surfactants include, e.g., perfluorooctanoate (PFOA orPFO), perfluorooctanesulfonate (PFOS), alkyl sulfate salts such assodium dodecyl sulfate (SDS) or ammonium lauryl sulfate, sodium laurethsulfate (also known as sodium lauryl ether sulfate, SLES), alkyl benzenesulfonate, and fatty acid salts. Exemplary cationic surfactants include,e.g., alkyltrimethylammonium salts such as cetyl trimethylammoniumbromide (CTAB, or hexadecyl trimethyl ammonium bromide), cetylpyridiniumchloride (CPC), polyethoxylated tallow amine (POEA), benzalkoniumchloride (BAC), benzethonium chloride (BZT). Exemplary zwitterionic(amphoteric) surfactants include, e.g., dodecyl betaine, cocamidopropylbetaine, and coco ampho glycinate. Exemplary nonionic surfactantsinclude, e.g., alkyl poly(ethylene oxide), alkylphenol poly(ethyleneoxide), copolymers of poly(ethylene oxide) and poly(propylene oxide)(commercially called poloxamers or poloxamines), Aayl polyglucosides(e.g., octyl glucoside or decyl maltoside), fatty alcohols (e.g., cetylalcohol or oleyl alcohol), cocamide MEA, cocamide DEA, Pluronic® F-68(polyoxyethylene-polyoxypropylene block copolymer), and polysorbates,such as Tween 20 (polysorbate 20), Tween 80 (polysorbate 80;polyoxyethylenesorbitan monooleate), dodecyl dimethylamine oxide, andvitamin E tocopherol propylene glycol succinate (Vitamin E TPGS).

A particularly useful group of surfactants are the sorbitan-basednon-ionic surfactants. These surfactants are prepared by dehydration ofsorbitol to give 1,4-sorbitan which is then reacted with one or moreequivalents of a fatty acid. The fatty-acid-substituted moiety may befurther reacted with ethylene oxide to give a second group ofsurfactants.

The fatty-acid-substituted sorbitan surfactants are made by reacting1,4-sorbitan with a fatty acid such as lauric acid, palmitic acid,stearic acid, oleic acid, or a similar long chain fatty acid to give the1,4-sorbitan mono-ester, 1,4-sorbitan sesquiester or 1,4-sorbitantriester. The common names for these surfactants include, for example,sorbitan monolaurate, sorbitan monopalmitate, sorbitan monoestearate,sorbitan monooleate, sorbitan sesquioleate, and sorbitan trioleate.These surfactants are commercially available under the name SPAN® orARLACEL®, usually with a letter or number designation whichdistinguishes between the various mono, di- and triester substitutedsorbitans.

SPAN® and ARLACEL® surfactants are hydrophilic and are generally solubleor dispersible in oil. They are also soluble in most organic solvents.In water they are generally insoluble but dispersible. Generally thesesurfactants will have a hydrophilic-lipophilic balance (HLB) numberbetween 1.8 to 8.6. Such surfactants can be readily made by means knownin the art or are commercially available.

A related group of surfactants comprises olyoxyethylene sorbitanmonoesters and olyoxyethylene sorbitan triesters. These materials areprepared by addition of ethylene oxide to a 1,4-sorbitan monester ortriester. The addition of polyoxyethylene converts the lipophilicsorbitan mono- or triester surfactant to a hydrophilic surfactantgenerally soluble or dispersible in water and soluble to varying degreesin organic liquids.

These materials, commercially available under the mark TWEEN®, areuseful for preparing oil-in-water emulsions and dispersions, or for thesolubilization of oils and making anhydrous ointments water-soluble orwashable. The TWEEN® surfactants may be combined with a related sorbitanmonester or triester surfactants to promote emulsion stability. TWEEN®surfactants generally have a HLB value falling between 9.6 to 16.7.TWEEN® surfactants are commercially available.

A third group of non-ionic surfactants which could be used alone or inconjunction with SPANS, ARLACEL® and TWEENS surfactants are thepolyoxyethylene fatty acids made by the reaction of ethylene oxide witha long-chain fatty acid. The most commonly available surfactant of thistype is solid under the name MYRJS and is a polyoxyethylene derivativeof stearic acid. MYRJ® surfactants are hydrophilic and soluble ordispersible in water like TWEEN® surfactants. The MYRJ® surfactants maybe blended with TWEEN® surfactants or with TWEEN®/SPAN® or ARLACEL®surfactant mixtures for use in forming emulsions. MYRJ® surfactants canbe made by methods known in the art or are available commercially.

A fourth group of polyoxyethylene based non-ionic surfactants are thepolyoxyethylene fatty acid ethers derived from lauryl, acetyl, stearyland oleyl alcohols. These materials are prepared as above by addition ofethylene oxide to a fatty alcohol. The commercial name for thesesurfactants is BRIJ®. BRIJ® surfactants may be hydrophilic or lipophilicdepending on the size of the polyoxyethylene moiety in the surfactant.While the preparation of these compounds is available from the art, theyare also readily available from commercial sources.

Other non-ionic surfactants which could potentially be used are, forexample, polyoxyethylene, polyol fatty acid esters, polyoxyethyleneether, polyoxypropylene fatty ethers, bee's wax derivatives containingpolyoxyethylene, polyoxyethylene lanolin derivative, polyoxyethylenefatty glycerides, glycerol fatty acid esters or other polyoxyethyleneacid alcohol or ether derivatives of long-chain fatty acids of 12-22carbon atoms.

As the emulsions and formulations of the invention are intended to bemulti-phase systems, it is preferable to choose an emulsion-formingnon-ionic surfactant which has an HLB value in the range of about 7 to16. This value may be obtained through the use of a single non-ionicsurfactant such as a TWEEN® surfactant or may be achieved by the use ofa blend of surfactants such as with a sorbitan mono, di- or triesterbased surfactant; a sorbitan ester polyoxyethylene fatty acid; asorbitan ester in combination with a polyoxyethylene lanolin derivedsurfactant; a sorbitan ester surfactant in combination with a high HLBpolyoxyethylene fatty ether surfactant; or a polyethylene fatty ethersurfactant or polyoxyethylene sorbitan fatty acid.

In certain embodiments, the emulsion comprises a single non-ionicsurfactant, most particularly a TWEEN® surfactant, as the emulsionstabilizing non-ionic surfactant. In an exemplary embodiment, theemulsion comprises TWEEN® 80, otherwise known as polysorbate 80 orpolyoxyethylene 20 sorbitan monooleate. In other embodiments, theemulsion comprises two or more non-ionic surfactants, in particular aTWEEN® surfactant and a SPAN® surfactant. In an exemplary embodiment,the emulsion comprises TWEEN® 80 and SPAN®85.

The oil-in-water emulsions can contain from about 0.01% to about 2.5%surfactant (v/v or w/v), about 0.01% to about 2% surfactant, 0.01% toabout 1.5% surfactant, 0.01% to about 1% surfactant, 0.01% to about 0.5%surfactant, 0.05% to about 0.5% surfactant, 0.08% to about 0.5%surfactant, about 0.08% surfactant, about 0.1% surfactant, about 0.2%surfactant, about 0.3% surfactant, about 0.4% surfactant, about 0.5%surfactant, about 0.6% surfactant, about 0.7% surfactant, about 0.8%surfactant, about 0.9% surfactant, or about 1% surfactant.

Alternatively or in addition, the oil-in-water emulsions can contain0.05% to about 1%, 0.05% to about 0.9%, 0.05% to about 0.8%, 0.05% toabout 0.7%, 0.05% to about 0.6%, 0.05% to about 0.5%, about 0.08%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, or about 1% Tween 80 (polysorbate 80;polyoxyethylenesorbitan monooleate).

In an exemplary embodiment, the oil-in-water emulsion contains 0.08%Tween 80.

Alternatively or in addition, the oil-in-water emulsions can contain0.05% to about 1%, 0.05% to about 0.9%, 0.05% to about 0.8%, 0.05% toabout 0.7%, 0.05% to about 0.6%, 0.05% to about 0.5%, about 0.08%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, or about 1% SPAN85 (sorbtian trioleate).

Alternatively or in addition, the oil-in-water emulsions can contain acombination of surfactants described herein. For example, a combinationof Tween 80 (polysorbate 80; polyoxyethylenesorbitan monooleate) andSPAN85 (sorbtian trioleate) may be used. The emulsions may containvarious amounts Tween 80 and SPAN85 (e.g., those exemplified above),including equal amounts of these surfactants. For example, theoil-in-water emulsions can contain about 0.05% Tween 80 and about 0.05%SPAN85, about 0.1% Tween 80 and about 0.1% SPAN85, about 0.2% Tween 80and about 0.2% SPAN85, about 0.3% Tween 80 and about 0.3% SPAN85, about0.4% Tween 80 and about 0.4% SPAN85, about 0.5% Tween 80 and about 0.5%SPAN85, about 0.6% Tween 80 and about 0.6% SPAN85, about 0.7% Tween 80and about 0.7% SPAN85, about 0.8% Tween 80 and about 0.8% SPAN85, about0.9% Tween 80 and about 0.9% SPAN85, or about 1% Tween 80 and about 1.0%SPAN85.

Polyethylene Glycol (PEG)-lipids, such as PEG coupled todialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG),PEG coupled to phosphatidylethanolamine (PE) (PEG-PE) or some otherphospholipids (PEG-phospholipids), PEG conjugated to ceramides(PEG-Cer), or a combination thereof, may also be used as surfactants(see, e.g., U.S. Pat. No. 5,885,613; U.S. patent application publicationNos. 2003/0077829, 2005/0175682 and 2006/0025366). Other suitablePEG-lipids include, e.g., PEG-dialkyloxypropyl (DAA) lipids orPEG-diacylglycerol (DAG) lipids. Exemplary PEG-DAG lipids include, e.g.,PEG-dilauroylglycerol (C₁₂) lipids, PEG-dimyristoylglycerol (C₁₄)lipids, PEG-dipalmitoylglycerol (C₁₆) lipids, or PEG-distearoylglycerol(C₁₈) lipids. Exemplary PEG-DAA lipids include, e.g.,PEG-dilauryloxypropyl (C₁₂) lipids, PEG-dimyristyloxypropyl (C₁₄)lipids, PEG-dipalmityloxypropyl (C₁₆) lipids, or PEG-distearyloxypropyl(C₁₈) lipids.

PEGs are classified by their molecular weights; for example, PEG 2000has an average molecular weight of about 2,000 daltons, and PEG 5000 hasan average molecular weight of about 5,000 daltons. PEGs arecommercially available from Sigma Chemical Co. as well as othercompanies and include, for example, the following:monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Inaddition, monomethoxypolyethyleneglycol-acetic acid (MePEG-CH₂COOH), isparticularly useful for preparing the PEG-lipid conjugates including,e.g., PEG-DAA conjugates.

Preferably, the PEG has an average molecular weight of from about 1000to about 5000 daltons (e.g., PEG₁₀₀₀, PEG₂₀₀₀, PEG₃₀₀₀, PEG₄₀₀₀,PEG₅₀₀₀). The PEG can be optionally substituted by an alkyl, alkoxy,acyl or aryl group. PEG can be conjugated directly to the lipid or maybe linked to the lipid via a linker moiety. Any linker moiety suitablefor coupling the PEG to a lipid can be used including, e.g., non-estercontaining linker moieties and ester-containing linker moieties

In exemplary embodiments, PEG₁₀₀₀PE, PEG₅₀₀₀PE, PEG₁₀₀₀DMG, PEG₂₀₀₀DMG,PEG₃₀₀₀DMG, or a combination thereof, is used as a surfactant. Incertain exemplary embodiments, the oil-in-water emulsion contains fromabout 1 mg/ml to about 80 mg/ml PEG₂₀₀₀PE, PEG₅₀₀₀PE, PEG₁₀₀₀DMG,PEG₂₀₀₀DMG, or PEG₃₀₀₀DMG.

Phospholipids

In certain embodiments, the particles of the cationic oil-in-wateremulsion further comprise a phospholipid.

Phospholipids are esters of fatty acids in which the alcohol componentof the molecule contains a phosphate group. Phospholipids includeglycerophosphatides (containing glycerol) and the sphingomyelins(containing sphingosine). Exemplary phospholipids includephosphatidylcholine, phosphatidylethanolamine, phosphatidylserine andsphingomyelin; and synthetic phospholipids comprising dimyristoylphosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoylphosphatidylcholine, distearoyl phosphatidylglycerol, dipalmitoylphosphatidylglycerol, dimyristoyl phosphatidylserine, distearoylphosphatidylserine, and dipalmitoyl serine.

The following exemplary phopholipids may be used.

DDPC 1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA-NA1,2-Dierucoyl-sn-Glycero-3-Phosphate(Sodium Salt) DEPC1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG-NA1,2-Dierucoyl-sn-Glycero-3 [Phosphatidyl-rac-(1- glycerol . . .) DLOPC1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA-NA1,2-Dilauroyl-sn-Glycero-3-Phosphate(Sodium Salt) DLPC1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine DLPG-NA1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .) (SodiumSalt) DLPG-NH4 1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol. . .) DLPS-NA 1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine(SodiumSalt) DMPA-NA 1,2-Diimyristoyl-sn-Glycero-3-Phosphate(Sodium Salt) DMPC1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine DMPG-NA1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .) DMPG-NH41,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .) DMPG-1,2-Myristoyl-sn-Glycero-3 [Phosphatidyl-rac-(1- NH4/NA glycerol . . .)DMPS-NA 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt)DOPA-NA 1,2-Dioleoyl-sn-Glycero-3-Phosphate(Sodium Salt) DOPC1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine DOPG-NA1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .) DOPS-NA1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt) DPPA-NA1,2-Dipalmitoyl-sn-Glycero-3-Phosphate(Sodium Salt) DPPC1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine DPPG-NA1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .)DPPG-NH4 1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . ..) DPPS-NA 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt)DPyPE 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA-NA1,2-Distearoyl-sn-Glycero-3-Phosphate(Sodium Salt) DSPC1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPE1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanolamine DSPG-NA1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .)DSPG-NH4 1,2-Distearoyl-sn-Glycero-3 [Phosphatidyl-rac-(1- glycerol . ..) DSPS-NA 1,2-Distearoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt)EPC Egg-PC HEPC Hydrogenated Egg PC HSPC High purity Hydrogenated Soy PCHSPC Hydrogenated Soy PC LYSOPC1-Myristoyl-sn-Glycero-3-phosphatidylcholine MYRISTIC LYSOPC1-Palmitoyl-sn-Glycero-3-phosphatidylcholine PALMITIC LYSOPC1-Stearoyl-sn-Glycero-3-phosphatidylcholine STEARIC Milk1-Myristoyl,2-palmitoyl-sn-Glycero 3- Sphingomyelin phosphatidylcholineMPPC MSPC 1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine PMPC1-Palmitoyl,2-myristoyl-sn-Glycero-3- phosphatidylcholine POPC1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine POPE1-Palmitoyl-2-oleoyl-sn-Glycero-3- phosphatidylethanolamine POPG-NA1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol) . . .](SodiumSalt) PSPC 1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine SMPC1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine SOPC1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine SPPC1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine

In certain embodiments, it may be advantageous to use a neutral lipid.It may also be advantageous to use a phospholipid, including azwitterionic phospholipid, for example, a phospholipid containing one ormore alkyl or alkenyl radicals of about 12 to about 22 carbons in length(e.g., about 12 to about 14, to about 16, to about 18, to about 20, toabout 22 carbons), which radicals may contain, for example, from 0 to 1to 2 to 3 double bonds. It may be advantageous to use a zwitterionicphospholipid.

Preferred phospholipids include, e.g.,1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), Eggphosphatidylcholine (egg PC), palmitoyl oleoyl phosphatidylcholine(POPC), dimyristoyl phosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), DPPC, dipalmitoyl phosphatidylcholine(DPPC), palmitoyl linoleyl phosphatidylcholine (PLPC), DPyPE, or acombination thereof.

In certain embodiments, the phospholipid is DOPE. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 20mg/ml DOPE. For example, the cationic oil-in-water emulsion may compriseDOPE at from about 0.5 mg/ml to about 10 mg/ml, from about 0.1 mg/ml toabout 10 mg/ml, or from about 1.5 mg/ml to about 7.5 mg/ml DOPE.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesabout 1.5 mg/ml DOPE.

In certain embodiments, the phospholipid is egg PC. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 20mg/ml egg PC. For example, the cationic oil-in-water emulsion maycomprise egg PC at from about 0.1 mg/ml to about 10 mg/ml, from about1.0 mg/ml to about 10 mg/ml, or from about 1.5 mg/ml to about 3.5 mg/mlegg PC.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesabout 1.55 mg/ml egg PC.

In certain embodiments, the phospholipid is DPyPE. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 20mg/ml DPyPE. For example, the cationic oil-in-water emulsion maycomprise DPyPE at from about 0.1 mg/ml to about 10 mg/ml, from about 1.5mg/ml to about 10 mg/ml, or from about 1.5 mg/ml to about 5 mg/ml DPyPE.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesabout 1.6 mg/ml DPyPE.

In certain embodiments, the emulsion particles may comprise acombination of a surfactant and a phospholipid described herein.

D. Aqueous Phase (Continuous Phase)

The aqueous phase (continuous phase) of the oil-in-water emulsions is abuffered salt solution (e.g., saline) or water. The buffered saltsolution is an aqueous solution that comprises a salt (e.g., NaCl), abuffer (e.g., a citrate buffer), and can further comprise an osmolalityadjusting agent (e.g., a saccharide), a polymer, a surfactant, or acombination thereof. If the emulsions are formulated for parenteraladministration, it is preferable to make up final buffered solutions sothat the tonicity, i.e., osmolality is essentially the same as normalphysiological fluids in order to prevent prevent undesiredpost-administration consequences, such as post-administration swellingor rapid absorption of the composition. It is also preferable to bufferthe aqueous phase in order to maintain a pH compatible with normalphysiological conditions. Also, in certain instances, it may bedesirable to maintain the pH at a particular level in order to insurethe stability of certain components of the emulsion.

For example, it may be desirable to prepare an emulsion that is isotonic(i.e., the same permeable solute (e.g., salt) concentration as thenormal cells of the body and the blood) and isosmotic. To controltonicity, the emulsion may comprise a physiological salt, such as asodium salt. Sodium chloride (NaCl), for example, may be used at about0.9% (w/v) (physiological saline). Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate, magnesium chloride, calcium chloride, etc. Non-ionictonicifying agents can also be used to control tonicity. A number ofnon-ionic tonicity modifying agents ordinarily known to those in theart. These are typically carbohydrates of various classifications (see,for example, Voet and Voet (1990) Biochemistry (John Wiley & Sons, NewYork). Monosaccharides classified as aldoses such as glucose, mannose,arabinose, and ribose, as well as those classified as ketoses such asfructose, sorbose, and xylulose can be used as non-ionic tonicifyingagents in the present invention. Disaccharides such a sucrose, maltose,trehalose, and lactose can also be used. In addition, alditols (acyclicpolyhydroxy alcohols, also referred to as sugar alcohols) such asglycerol, mannitol, xylitol, and sorbitol are non-ionic tonicifyingagents useful in the present invention. Non-ionic tonicity modifyingagents can be present at a concentration of from about 0.1% to about 10%or about 1% to about 10%, depending upon the agent that is used.

The aqueous phase may be buffered. Any physiologically acceptable buffermay be used herein, such as water, citrate buffers, phosphate buffers,acetate buffers, tris buffers, bicarbonate buffers, carbonate buffers,succinate buffer, or the like. The pH of the aqueous component willpreferably be between 6.0-8.0, preferably about 6.2 to about 6.8. In anexemplary embodiment, the buffer is 10 mM citrate buffer with a pH at6.5. In another exemplary embodiment, the aqueous phase is, or thebuffer prepared using, RNase-free water or DEPC treated water. In somecases, high salt in the buffer might interfere with complexation ofnegatively charged molecule to the emulsion particle therefore isavoided. In other cases, certain amount of salt in the buffer may beincluded.

In an exemplary embodiment, the buffer is 10 mM citrate buffer with a pHat 6.5. In another exemplary embodiment, the aqueous phase is, or thebuffer is prepared using, RNase-free water or DEPC treated water.

The aqueous phase may also comprise additional components such asmolecules that change the osmolarity of the aqueous phase or moleculesthat stabilizes the negatively charged molecule after complexation.Preferably, the osmolarity of the aqueous phase is adjusting using anon-ionic tonicifying agent, such as a sugar (e.g., trehalose, sucrose,dextrose, fructose, reduced palatinose, etc.), a sugar alcohol (such asmannitol, sorbitol, xylitol, erythritol, lactitol, maltitol, glycerol,etc.), or combinations thereof. If desired, a nonionic polymer (e.g., apoly(alkyl glycol) such as polyethylene glycol, polypropylene glycol, orpolybutlyene glycol) or nonionic surfactant can be used.

In some case, unadulterated water may be preferred as the aqueous phaseof the emulsion when the emulsion is initially prepared. For example,increasing the salt concentration may make it more difficult to achievethe desirable particle size (e.g., less than about 200 nm).

In certain embodiments, the aqueous phase of the cationic oil-in-wateremulsion may further comprise a polymer or a surfactant, or acombination thereof. In an exemplary embodiment, the oil-in-wateremulsion contains a poloxamer. Poloxamers are nonionic triblockcopolymers having a central hydrophobic chain of polyoxypropylene(poly(propylene oxide)) flanked by two hydrophilic chains ofpolyoxyethylene (poly(ethylene oxide)). Poloxamers are also known by thetrade name Pluronic® polymers. Poloxamer polymers may lead to greaterstability and increased RNase resistance of the RNA molecule after RNAcomplexation.

Alternatively or in addition, the cationic oil-in-water emulsion maycomprise from about 0.1% to about 20% (w/v) polymer, or from about 0.05%to about 10% (w/v) polymer. For example, the cationic oil-in-wateremulsion may comprise a polymer (e.g., a poloxamer such as Pluronic®F127) at from about 0.1% to about 20% (w/v), from about 0.1% to about10% (w/v), from about 0.05% to about 10% (w/v), or from about 0.05% toabout 5% (w/v).

In an exemplary embodiment, the oil-in-water emulsion comprises about 4%(w/v), or about 8% (w/v) Pluronic® F127.

The quantity of the aqueous component employed in these compositionswill be that amount necessary to bring the value of the composition tounity. That is, a quantity of aqueous component sufficient to make 100%will be mixed, with the other components listed above in order to bringthe compositions to volume.

4. Negatively Charged Molecules

When a negatively charged molecule is to be delivered, it can becomplexed with the particles of the cationic oil-in-water emulsions. Thenegatively charged molecule is complexed with the emulsion particles by,for example, interactions between the negatively charged molecule andthe cationic lipid on the surface of the particles, as well ashydrophobic/hydrophilic interactions between the negatively chargedmolecule and the surface of the particles. Although not wishing to bebound by any particular theory, it is believed that the negativelycharged molecules interact with the cationic lipid through non-covalent,ionic charge interactions (electrostatic forces), and the strength ofthe complex as well as the amount of negatively charged compound thatcan be complexed to a particle are related to the amount of cationiclipid in the particle. Additionally, hydrophobic/hydrophilicinteractions between the negatively charged molecule and the surface ofthe particles may also play a role.

Examples of negatively charged molecules include negatively chargedpeptides, polypeptides or proteins, nucleic acid molecules (e.g., singleor double stranded RNA or DNA), small molecules (e.g., small moleculeimmune potentiators (SMIPs), phosphonate, fluorophosphonate, etc.) andthe like. In preferred aspects, the negatively charged molecule is anRNA molecule, such as an RNA that encodes a peptide, polypeptide orprotein, including self-replicating RNA molecules, or a smallinterfering RNA.

The complex can be formed by using techniques known in the art, examplesof which are described herein. For example, a nucleic acid-particlecomplex can be formed by mixing a cationic emulsion with the nucleicacid molecule, for example by vortexing. The amount of the negativelycharged molecule and cationic lipid in the emulsions may be adjusted oroptimized to provide desired strength of binding and binding capacity.

For example, as described and exampled herein, exemplary RNA-particlecomplexes were produced by varying the RNA: cationic lipid ratios (asmeasured by the “N/P ratio”). The term N/P ratio refers to the amount(moles) of protonatable nitrogen atoms in the cationic lipid divided bythe amount (moles) of phosphates on the RNA. Preferred N/P ratios arefrom about 1:1 to about 20:1, from about 2:1 to about 18:1, from about3:1 to 16:1, from about 4:1 to about 14:1, from about 6:1 to about 12:1,about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about15:1, or about 16:1. Alternatively, preferred N/P ratios are at leastabout 3:1, at least about 4:1, at least about 5:1, at least about 6:1,at least about 7:1, at least about 8:1, at least about 9:1, at leastabout 10:1, at least about 11:1, at least about 12:1, at least about13:1, at least about 14:1, at least about 15:1, or at least about 16:1.A more preferred N/P ratio is about 4:1 or higher.

Each emulsion may have its own optimal or preferred N/P ratio to producedesired effects (e.g., desired level of expression of the complexedRNA), which can be determined experimentally (e.g., using the assays asdescribed herein or other techniques known in the art, such as measuringexpression level of a protein that is encoded by the RNA, or measuringthe percentage of the RNA molecules being released from the complex inthe presence of heparin). Generally, the N/P ratio should be at a valuethat at least about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,or about 95% of the RNA molecules are released from the RNA-particlecomplexes when the RNA-particle complexes are taken up by cells. An N/Pratio of at least 4:1 is preferred.

The cationic oil-in-water emulsions described herein are particularlysuitable for formulating nucleic acid-based vaccines (e.g., DNAvaccines, RNA vaccines). The formation of a nucleic acid-emulsionparticle complex facilitates the uptake of the nucleic acid into hostcells, and protects the nucleic acid molecule from nuclease degradation.Transfected cells can then express the antigen encoded by the nucleicacid molecule, which can produce an immune response to the antigen. Likelive or attenuated viruses, nucleic acid-based vaccines can effectivelyengage both MHC-I and MHC-II pathways allowing for the induction of CD8⁺and CD4⁺ T cell responses, whereas antigen present in soluble form, suchas recombinant protein, generally induces only antibody responses.

The sequence of the RNA molecule may be codon optimized or deoptimizedfor expression in a desired host, such as a human cell.

In certain embodiments, the negatively charged molecule described hereinis an RNA molecule. In certain embodiments, the RNA molecule encodes anantigen (peptide, polypeptide or protein) and the cationic oil in wateremulsion is suitable for use as an RNA-based vaccine. The compositioncan contain more than one RNA molecule encoding an antigen, e.g., two,three, five, or ten RNA molecules that are complexed to the emulsionparticles. That is, the composition can contain one or more differentspecies of RNA molecules, each encoding a different antigen.Alternatively or in addition, one RNA molecule may also encode more thanone antigen, e.g., a bicistronic, or tricistronic RNA molecule thatencodes different or identical antigens. Accordingly, the cationic oilin water emulsion is suitable for use as an RNA-based vaccine, that ismonovalent or multivalent.

The sequence of the RNA molecule may be modified if desired, for exampleto increase the efficacy of expression or replication of the RNA, or toprovide additional stability or resistance to degradation. For example,the RNA sequence can be modified with respect to its codon usage, forexample, to increase translation efficacy and half-life of the RNA. Apoly A tail (e.g., of about 30 adenosine residues or more) may beattached to the 3′ end of the RNA to increase its half-life. The 5′ endof the RNA may be capped with a modified ribonucleotide with thestructure m7G (5′) ppp (5′) N (cap 0 structure) or a derivative thereof,which can be incorporated during RNA synthesis or can be enzymaticallyengineered after RNA transcription (e.g., by using Vaccinia VirusCapping Enzyme (VCE) consisting of mRNA triphosphatase,guanylyl-transferase and guanine-7-methylransferase, which catalyzes theconstruction of N7-monomethylated cap 0 structures). Cap 0 structureplays an important role in maintaining the stability and translationalefficacy of the RNA molecule. The 5′ cap of the RNA molecule may befurther modified by a 2′-O-Methyltransferase which results in thegeneration of a cap 1 structure (m7 Gppp[m2′-O]N), which may furtherincreases translation efficacy.

If desired, the RNA molecule can comprise one or more modifiednucleotides. This can be in addition to any 5′ cap structure. There aremore than 96 naturally occurring nucleoside modifications found onmammalian RNA. See, e.g., Limbach et al., Nucleic Acids Research,22(12):2183-2196 (1994). The preparation of nucleotides and modifiednucleotides and nucleosides are well-known in the art, e.g. from U.S.Pat. Nos. 4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679,5,047,524, 5,132,418, 5,153,319, 5,262,530, 5,700,642 all of which areincorporated by reference in their entirety herein, and many modifiednucleosides and modified nucleotides are commercially available.

Modified nucleobases which can be incorporated into modified nucleosidesand nucleotides and be present in the RNA molecules include: m5C(5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U(2-thiouridine), Um (2′-O-methyluridine), m1A (1-methyladenosine); m2A(2-methyladenosine); Am (2-1-O-methyladenosine); ms2 m6A(2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2m6A(2-methylthio-N6 isopentenyladenosine); io6A(N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine);ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m1I (1-methylinosine);m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm(2T-O-methylcytidine); s2C (2-thiocytidine); ac4C(N-4-acetylcytidine);f5C (5-formylcytidine); m5Cm (5,2-O-dimethyl cytidine); ac4Cm(N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine);m2G (N-2-methylguanosine); m7G (7-methylguanosine); Gm(2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm(N2,2′-O-dimethylguanosine); m22Gm (N²,N2,2′-β-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q(queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi(7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine);mSUm (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U(5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm (2′-O-methylinosine);m4C(N-4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hmSC(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U(5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am(N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G(N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5 Cm (5-formyl-2′-O-methylcytidine); m1Gm(1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine)irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14(4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine),hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof,dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-(C₁-C₆)-alkyluracil, 5-methyluracil, 5-(C₂-C₆)-alkenyluracil,5-(C₂-C₆)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil,5-fluorouracil, 5-bromouracil, 5-hydroxycytosine,5-(C₁-C₆)-alkylcytosine, 5-methylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, dimethylguanine, 7-deazaguanine, 8-azaguanine,7-deaza-7-substituted guanine, 7-deaza-7-(C₂-C₆)alkynylguanine,7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine,8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine,2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine,7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen(abasic residue), mSC, mSU, m6A, s2U, W, or 2′-O-methyl-U. Many of thesemodified nucleobases and their corresponding ribonucleosides areavailable from commercial suppliers. See, e.g., WO 2011/005799 which isincorporated herein by reference.

A RNA used with the invention ideally includes only phosphodiesterlinkages between nucleosides, but in some embodiments it can containphosphoramidate, phosphorothioate, and/or methylphosphonate linkages.

In some embodiments, the RNA molecule does not include modifiednucleotides, e.g., does not include modified nucleobases, and all of thenucleotides in the RNA molecule are conventional standardribonucleotides A, U, G and C, with the exception of an optional 5′ capthat may include, for example, 7-methylguanosine. In other embodiments,the RNA may include a 5′ cap comprising a 7′-methylguanosine, and thefirst 1, 2 or 3 5′ ribonucleotides may be methylated at the 2′ positionof the ribose.

A. Self-Replicating RNA

In some aspects, the cationic oil in water emulsion contains aself-replicating RNA molecule. In certain embodiments, theself-replicating RNA molecule is derived from or based on an alphavirus.

Self-replicating RNA molecules are well known in the art and can beproduced by using replication elements derived from, e.g., alphaviruses,and substituting the structural viral proteins with a nucleotidesequence encoding a protein of interest. A self-replicating RNA moleculeis typically a (+)-strand molecule which can be directly translatedafter delivery to a cell, and this translation provides a RNA-dependentRNA polymerase which then produces both antisense and sense transcriptsfrom the delivered RNA. Thus the delivered RNA leads to the productionof multiple daughter RNAs. These daughter RNAs, as well as collinearsubgenomic transcripts, may be translated themselves to provide in situexpression of an encoded antigen, or may be transcribed to providefurther transcripts with the same sense as the delivered RNA which aretranslated to provide in situ expression of the antigen. The overallresults of this sequence of transcriptions is a huge amplification inthe number of the introduced replicon RNAs and so the encoded antigenbecomes a major polypeptide product of the cells. Cells transfected withself-replicating RNA briefly produce of antigen before undergoingapoptotic death. This death is a likely result of requisitedouble-stranded (ds) RNA intermediates, which also have been shown tosuper-activate Dendritic Cells. Thus, the enhanced immunogenicity ofself-replicating RNA may be a result of the production ofpro-inflammatory dsRNA, which mimics an RNA-virus infection of hostcells.

Advantageously, the cell's machinery is used by self-replicating RNAmolecules to generate an exponential increase of encoded gene products,such as proteins or antigens, which can accumulate in the cells or besecreted from the cells. Overexpression of proteins by self-replicatingRNA molecules takes advantage of the immunostimulatory adjuvant effects,including stimulation of toll-like receptors (TLR) 3, 7 and 8 and nonTLR pathways (e.g, RIG-1, MD-5) by the products of RNA replication andamplification, and translation which induces apoptosis of thetransfected cell.

The self-replicating RNA generally contains at least one or more genesselected from the group consisting of viral replicases, viral proteases,viral helicases and other nonstructural viral proteins, and alsocomprise 5′- and 3′-end cis-active replication sequences, and ifdesired, a heterologous sequences that encode a desired amino acidsequences (e.g., an antigen of interest). A subgenomic promoter thatdirects expression of the heterologous sequence can be included in theself-replicating RNA. If desired, the heterologous sequence (e.g., anantigen of interest) may be fused in frame to other coding regions inthe self-replicating RNA and/or may be under the control of an internalribosome entry site (IRES).

In certain embodiments, the self-replicating RNA molecule is notencapsulated in a virus-like particle. Self-replicating RNA molecules ofthe invention can be designed so that the self-replicating RNA moleculecannot induce production of infectious viral particles. This can beachieved, for example, by omitting one or more viral genes encodingstructural proteins that are necessary for the production of viralparticles in the self-replicating RNA. For example, when theself-replicating RNA molecule is based on an alpha virus, such asSinebis virus (SIN), Semliki forest virus and Venezuelan equineencephalitis virus (VEE), one or more genes encoding viral structuralproteins, such as capsid and/or envelope glycoproteins, can be omitted.

If desired, self-replicating RNA molecules of the invention can also bedesigned to induce production of infectious viral particles that areattenuated or virulent, or to produce viral particles that are capableof a single round of subsequent infection.

One suitable system for achieving self-replication in this manner is touse an alphavirus-based replicon. Alphaviruses comprise a set ofgenetically, structurally, and serologically related arthropod-borneviruses of the Togaviridae family. Twenty-six known viruses and virussubtypes have been classified within the alphavirus genus, including,Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelanequine encephalitis virus. As such, the self-replicating RNA of theinvention may incorporate a RNA replicase derived from semliki forestvirus (SFV), sindbis virus (SIN), Venezuelan equine encephalitis virus(VEE), Ross-River virus (RRV), eastern equine encephalitis virus, orother viruses belonging to the alphavirus family.

An alphavirus-based “replicon” expression vectors can be used in theinvention. Replicon vectors may be utilized in several formats,including DNA, RNA, and recombinant replicon particles. Such repliconvectors have been derived from alphaviruses that include, for example,Sindbis virus (Xiong et al. (1989) Science 243:1188-1191; Dubensky etal., (1996) J. Virol. 70:508-519; Hariharan et al. (1998) J. Virol.72:950-958; Polo et al. (1999) PNAS 96:4598-4603), Semliki Forest virus(Liljestrom (1991) Bio/Technology 9:1356-1361; Berglund et al. (1998)Nat. Biotech. 16:562-565), and Venezuelan equine encephalitis virus(Pushko et al. (1997) Virology 239:389-401). Alphaviruses-derivedreplicons are generally quite similar in overall characteristics (e.g.,structure, replication), individual alphaviruses may exhibit someparticular property (e.g., receptor binding, interferon sensitivity, anddisease profile) that is unique. Therefore, chimeric alphavirusreplicons made from divergent virus families may also be useful.

Alphavirus-based RNA replicons are typically (+)-stranded RNAs whichlead to translation of a replicase (or replicase-transcriptase) afterdelivery to a cell. The replicase is translated as a polyprotein whichauto-cleaves to provide a replication complex which creates genomic(−)-strand copies of the (+)-strand delivered RNA. These (−)-strandtranscripts can themselves be transcribed to give further copies of the(+)-stranded parent RNA and also to give a subgenomic transcript whichencodes the antigen. Translation of the subgenomic transcript thus leadsto in situ expression of the antigen by the infected cell. Suitablealphavirus replicons can use a replicase from a Sindbis virus, a Semlikiforest virus, an eastern equine encephalitis virus, a Venezuelan equineencephalitis virus, etc.

An RNA replicon preferably comprises an RNA genome from a picornavirus,togavirus, flavivirus, coronavirus, paramyxovirus, yellow fever virus,or alphavirus (e.g., Sindbis virus, Semliki Forest virus, Venezuelanequine encephalitis virus, or Ross River virus), which has been modifiedby the replacement of one or more structural protein genes with aselected heterologous nucleic acid sequence encoding a product ofinterest.

A preferred replicon encodes (i) a RNA-dependent RNA polymerase whichcan transcribe RNA from the replicon and (ii) an antigen. The polymerasecan be an alphavirus replicase e.g. comprising one or more of alphavirusproteins nsP1, nsP2, nsP3 and nsP4. Whereas natural alphavirus genomesencode structural virion proteins in addition to the non-structuralreplicase polyprotein, it is preferred that the replicon does not encodealphavirus structural proteins. Thus a preferred replicon can lead tothe production of genomic RNA copies of itself in a cell, but not to theproduction of RNA-containing virions. The inability to produce thesevirions means that, unlike a wild-type alphavirus, the preferredreplicon cannot perpetuate itself in infectious form. The alphavirusstructural proteins which are necessary for perpetuation in wild-typeviruses are absent from the preferred replicon and their place is takenby gene(s) encoding the antigen of interest, such that the subgenomictranscript encodes the antigen rather than the structural alphavirusvirion proteins.

A replicon useful with the invention may have two open reading frames.The first (5′) open reading frame encodes a replicase; the second (3′)open reading frame encodes an antigen. In some embodiments the RNA mayhave additional (e.g. downstream) open reading frames e.g. to encodeadditional antigens or to encode accessory polypeptides.

A preferred replicon has a 5′ cap (e.g. a 7-methylguanosine), whichoften can enhance in vivo translation of the RNA. In some embodimentsthe 5′ sequence of the replicon may need to be selected to ensurecompatibility with the encoded replicase.

A replicon may have a 3′ poly-A tail. It may also include a poly-Apolymerase recognition sequence (e.g. AAUAAA) near its 3′ end.

Replicons can have various lengths but they are typically 5000-25000nucleotides long e.g. 8000-15000 nucleotides, or 9000-12000 nucleotides.

The replicon can conveniently be prepared by in vitro transcription(IVT). IVT can use a (cDNA) template created and propagated in plasmidform in bacteria, or created synthetically (for example by genesynthesis and/or polymerase chain-reaction (PCR) engineering methods).For instance, a DNA-dependent RNA polymerase (such as the bacteriophageT7, T3 or SP6 RNA polymerases) can be used to transcribe the repliconfrom a DNA template. Appropriate capping and poly-A addition reactionscan be used as required (although the replicon's poly-A is usuallyencoded within the DNA template). These RNA polymerases can havestringent requirements for the transcribed 5′ nucleotide(s) and in someembodiments these requirements must be matched with the requirements ofthe encoded replicase, to ensure that the IVT-transcribed RNA canfunction efficiently as a substrate for its self-encoded replicase.Specific examples include Sindbis-virus-based plasmids (pSIN) such aspSINCP, described, for example, in U.S. Pat. Nos. 5,814,482 and6,015,686, as well as in International Publication Nos. WO 97/38087, WO99/18226 and WO 02/26209. The construction of such replicons, ingeneral, is described in U.S. Pat. Nos. 5,814,482 and 6,015,686.

In other aspects, the self-replicating RNA molecule is derived from orbased on a virus other than an alphavirus, preferably, apositive-stranded RNA virus, and more preferably a picornavirus,flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, orcoronavirus. Suitable wild-type alphavirus sequences are well-known andare available from sequence depositories, such as the American TypeCulture Collection, Rockville, Md. Representative examples of suitablealphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCCVR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCCVR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCCVR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCCVR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus(ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580,ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCCVR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest(ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248),Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374),Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCCVR-1250 ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis(ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCCVR-926), and Y-62-33 (ATCC VR-375).

The self-replicating RNA molecules of the invention are larger thanother types of RNA (e.g. mRNA) that have been prepared using modifiednucleotides. Typically, the self-replicating RNA molecules of theinvention contain at least about 4 kb. For example, the self-replicatingRNA can contain at least about 5 kb, at least about 6 kb, at least about7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, atleast about 11 kb, at least about 12 kb or more than 12 kb. In certainexamples, the self-replicating RNA is about 4 kb to about 12 kb, about 5kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb,about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb toabout 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb,about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb toabout 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb,about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb toabout 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb,about 9 kb to about 10 kb, or about 10 kb to about 11 kb.

The self-replicating RNA molecules of the invention may comprise one ormore types of modified nucleotides (e.g., pseudouridine,N6-methyladenosine, 5-methylcytidine, 5-methyluridine).

The self-replicating RNA molecule may encode a single heterologouspolypeptide antigen or, optionally, two or more heterologous polypeptideantigens linked together in a way that each of the sequences retains itsidentity (e.g., linked in series) when expressed as an amino acidsequence. The heterologous polypeptides generated from theself-replicating RNA may then be produced as a fusion polypeptide orengineered in such a manner to result in separate polypeptide or peptidesequences.

The self-replicating RNA of the invention may encode one or morepolypeptide antigens that contain a range of epitopes. Preferablyepitopes capable of eliciting either a helper T-cell response or acytotoxic T-cell response or both.

The self-replicating RNA molecules described herein may be engineered toexpress multiple nucleotide sequences, from two or more open readingframes, thereby allowing co-expression of proteins, such as a two ormore antigens together with cytokines or other immunomodulators, whichcan enhance the generation of an immune response. Such aself-replicating RNA molecule might be particularly useful, for example,in the production of various gene products (e.g., proteins) at the sametime, for example, as a bivalent or multivalent vaccine.

The self-replicating RNA molecules of the invention can be preparedusing any suitable method. Several suitable methods are known in the artfor producing RNA molecules that contain modified nucleotides. Forexample, a self-replicating RNA molecule that contains modifiednucleotides can be prepared by transcribing (e.g., in vitrotranscription) a DNA that encodes the self-replicating RNA moleculeusing a suitable DNA-dependent RNA polymerase, such as T7 phage RNApolymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, and thelike, or mutants of these polymerases which allow efficientincorporation of modified nucleotides into RNA molecules. Thetranscription reaction will contain nucleotides and modifiednucleotides, and other components that support the activity of theselected polymerase, such as a suitable buffer, and suitable salts. Theincorporation of nucleotide analogs into a self-replicating RNA may beengineered, for example, to alter the stability of such RNA molecules,to increase resistance against RNases, to establish replication afterintroduction into appropriate host cells (“infectivity” of the RNA),and/or to induce or reduce innate and adaptive immune responses.

Suitable synthetic methods can be used alone, or in combination with oneor more other methods (e.g., recombinant DNA or RNA technology), toproduce a self-replicating RNA molecule of the invention. Suitablemethods for de novo synthesis are well-known in the art and can beadapted for particular applications. Exemplary methods include, forexample, chemical synthesis using suitable protecting groups such as CEM(Masuda et al., (2007) Nucleic Acids Symposium Series 51:3-4), theβ-cyanoethyl phosphoramidite method (Beaucage S L et al. (1981)Tetrahedron Lett 22:1859); nucleoside H-phosphonate method (Garegg P etal. (1986) Tetrahedron Lett 27:4051-4; Froehler B C et al. (1986) NuclAcid Res 14:5399-407; Garegg P et al. (1986) Tetrahedron Lett 27:4055-8;Gaffney B L et al. (1988) Tetrahedron Lett 29:2619-22). Thesechemistries can be performed or adapted for use with automated nucleicacid synthesizers that are commercially available. Additional suitablesynthetic methods are disclosed in Uhlmann et al. (1990) Chem Rev90:544-84, and Goodchild J (1990) Bioconjugate Chem 1: 165. Nucleic acidsynthesis can also be performed using suitable recombinant methods thatare well-known and conventional in the art, including cloning,processing, and/or expression of polynucleotides and gene productsencoded by such polynucleotides. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic polynucleotides areexamples of known techniques that can be used to design and engineerpolynucleotide sequences. Site-directed mutagenesis can be used to alternucleic acids and the encoded proteins, for example, to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, introduce mutations and the like.Suitable methods for transcription, translation and expression ofnucleic acid sequences are known and conventional in the art. (Seegenerally, Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel,et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988;Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986;Bitter, et al., in Methods in Enzymology 153:516-544 (1987); TheMolecular Biology of the Yeast Saccharomyces, Eds. Strathern et al.,Cold Spring Harbor Press, Vols. I and II, 1982; and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989.)

The presence and/or quantity of one or more modified nucleotides in aself-replicating RNA molecule can be determined using any suitablemethod. For example, a self-replicating RNA can be digested tomonophosphates (e.g., using nuclease P1) and dephosphorylated (e.g.,using a suitable phosphatase such as CIAP), and the resultingnucleosides analyzed by reversed phase HPLC (e.g., usings a YMC PackODS-AQ column (5 micron, 4.6×250 mm) and elute using a gradient, 30% B(0-5 min) to 100% B (5-13 min) and at 100% B (13-40) min, flow Rate (0.7ml/min), UV detection (wavelength: 260 nm), column temperature (30° C.).Buffer A (20 mM acetic acid-ammonium acetate pH 3.5), buffer B (20 mMacetic acid-ammonium acetate pH 3.5/methanol [90/10])).

Optionally, the self-replicating RNA molecules of the invention mayinclude one or more modified nucleotides so that the self-replicatingRNA molecule will have less immunomodulatory activity upon introductionor entry into a host cell (e.g., a human cell) in comparison to thecorresponding self-replicating RNA molecule that does not containmodified nucleotides.

If desired, the self-replicating RNA molecules can be screened oranalyzed to confirm their therapeutic and prophylactic properties usingvarious in vitro or in vivo testing methods that are known to those ofskill in the art. For example, vaccines comprising self-replicating RNAmolecule can be tested for their effect on induction of proliferation oreffector function of the particular lymphocyte type of interest, e.g., Bcells, T cells, T cell lines, and T cell clones. For example, spleencells from immunized mice can be isolated and the capacity of cytotoxicT lymphocytes to lyse autologous target cells that contain a selfreplicating RNA molecule that encodes a polypeptide antigen. Inaddition, T helper cell differentiation can be analyzed by measuringproliferation or production of TH1 (IL-2 and IFN-γ) and/or TH2 (IL-4 andIL-5) cytokines by ELISA or directly in CD4+ T cells by cytoplasmiccytokine staining and flow cytometry.

Self-replicating RNA molecules that encode a polypeptide antigen canalso be tested for ability to induce humoral immune responses, asevidenced, for example, by induction of B cell production of antibodiesspecific for an antigen of interest. These assays can be conductedusing, for example, peripheral B lymphocytes from immunized individuals.Such assay methods are known to those of skill in the art. Other assaysthat can be used to characterize the self-replicating RNA molecules ofthe invention can involve detecting expression of the encoded antigen bythe target cells. For example, FACS can be used to detect antigenexpression on the cell surface or intracellularly. Another advantage ofFACS selection is that one can sort for different levels of expression;sometimes-lower expression may be desired. Other suitable method foridentifying cells which express a particular antigen involve panningusing monoclonal antibodies on a plate or capture using magnetic beadscoated with monoclonal antibodies.

B. Antigens

In certain embodiments, the negatively charged molecule described hereinis a nucleic acid molecule (e.g., an RNA molecule) that encodes anantigen. Suitable antigens include, but are not limited to, a bactertialantigen, a viral antigen, a fungal antigen, a protazoan antigen, a plantantigen, a cancer antigen, or a combination thereof.

Suitable antigens include proteins and peptides from a pathogen such asa virus, bacteria, fungus, protozoan, plant or from a tumor. Viralantigens and immunogens that can be encoded by the self-replicating RNAmolecule include, but are not limited to, proteins and peptides from aOrthomyxoviruses, such as Influenza A, B and C; Paramyxoviridae viruses,such as Pneumoviruses (RSV), Paramyxoviruses (PIV), Metapneumovirus andMorbilliviruses (e.g., measles); Pneumoviruses, such as Respiratorysyncytial virus (RSV), Bovine respiratory syncytial virus, Pneumoniavirus of mice, and Turkey rhinotracheitis virus; Paramyxoviruses, suchas Parainfluenza virus types 1-4 (PIV), Mumps virus, Sendai viruses,Simian virus 5, Bovine parainfluenza virus, Nipahvirus, Henipavirus andNewcastle disease virus; Poxyiridae, including a Orthopoxvirus such asVariola vera (including but not limited to, Variola major and Variolaminor); Metapneumoviruses, such as human metapneumovirus (hMPV) andavian metapneumoviruses (aMPV); Morbilliviruses, such as Measles;Picornaviruses, such as Enteroviruses, Rhinoviruses, Heparnavirus,Parechovirus, Cardioviruses and Aphthoviruses; Enteroviruseses, such asPoliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24,Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus types 1 to 9, 11to 27 and 29 to 34 and Enterovirus 68 to 71, Bunyaviruses, including aOrthobunyavirus such as California encephalitis virus; a Phlebovirus,such as Rift Valley Fever virus; a Nairovirus, such as Crimean-Congohemorrhagic fever virus; Heparnaviruses, such as, Hepatitis A virus(HAV); Togaviruses (Rubella), such as a Rubivirus, an Alphavirus, or anArterivirus; Flaviviruses, such as Tick-borne encephalitis (TBE) virus,Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japaneseencephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus,St. Louis encephalitis virus, Russian spring-summer encephalitis virus,Powassan encephalitis virus; Pestiviruses, such as Bovine viral diarrhea(BVDV), Classical swine fever (CSFV) or Border disease (BDV);Hepadnaviruses, such as Hepatitis B virus, Hepatitis C virus;Rhabdoviruses, such as a Lyssavirus (Rabies virus) and Vesiculovirus(VSV), Caliciviridae, such as Norwalk virus, and Norwalk-like Viruses,such as Hawaii Virus and Snow Mountain Virus; Coronaviruses, such asSARS, Human respiratory coronavirus, Avian infectious bronchitis (IBV),Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritisvirus (TGEV); Retroviruses such as an Oncovirus, a Lentivirus or aSpumavirus; Reoviruses, as an Orthoreovirus, a Rotavirus, an Orbivirus,or a Coltivirus; Parvoviruses, such as Parvovirus B19; Delta hepatitisvirus (HDV); Hepatitis E virus (HEV); Hepatitis G virus (HGV); HumanHerpesviruses, such as, by way Herpes Simplex Viruses (HSV),Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus(CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and HumanHerpesvirus 8 (HHV8); Papovaviruses, such as Papillomaviruses andPolyomaviruses, Adenoviruess and Arenaviruses.

In some embodiments, the antigen elicits an immune response against avirus which infects fish, such as: infectious salmon anemia virus(ISAV), salmon pancreatic disease virus (SPDV), infectious pancreaticnecrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystisdisease virus (FLDV), infectious hematopoietic necrosis virus (IHNV),koi herpesvirus, salmon picorna-like virus (also known as picorna-likevirus of atlantic salmon), landlocked salmon virus (LSV), atlanticsalmon rotavirus (ASR), trout strawberry disease virus (TSD), cohosalmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).

In some embodiments the antigen elicits an immune response against aparasite from the Plasmodium genus, such as P. falciparum, P. vivax, P.malariae or P. ovale. Thus the invention may be used for immunisingagainst malaria. In some embodiments the antigen elicits an immuneresponse against a parasite from the Caligidae family, particularlythose from the Lepeophtheirus and Caligus genera e.g. sea lice such asLepeophtheirus salmonis or Caligus rogercresseyi.

Bacterial antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Neisseria meningitides, Streptococcus pneumoniae,Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis,Burkholderia sp. (e.g., Burkholderia mallei, Burkholderia pseudomalleiand Burkholderia cepacia), Staphylococcus aureus, Staphylococcusepidermis, Haemophilus influenzae, Clostridium tetani (Tetanus),Clostridium perfringens, Clostridium botulinums (Botulism),Cornynebacterium diphtheriae (Diphtheria), Pseudomonas aeruginosa,Legionella pneumophila, Coxiella burnetii, Brucella sp. (e.g., B.abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis and B.pinnipediae,), Francisella sp. (e.g., F. novicida, F. philomiragia andF. tularensis), Streptococcus agalactiae, Neiserria gonorrhoeae,Chlamydia trachomatis, Treponema pallidum (Syphilis), Haemophilusducreyi, Enterococcus faecalis, Enterococcus faecium, Helicobacterpylori, Staphylococcus saprophyticus, Yersinia enterocolitica, E. coli(such as enterotoxigenic E. coli (ETEC), enteroaggregative E. coli(EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli(EPEC), extraintestinal pathogenic E. coli (ExPEC; such as uropathogenicE. coli (UPEC) and meningitis/sepsis-associated E. coli (MNEC)), and/orenterohemorrhagic E. coli (EHEC), Bacillus anthracis (anthrax), Yersiniapestis (plague), Mycobacterium tuberculosis, Rickettsia, Listeriamonocytogenes, Chlamydia pneumoniae, Vibrio cholerae, Salmonella typhi(typhoid fever), Borrelia burgdorfer, Porphyromonas gingivalis,Klebsiella, Mycoplasma pneumoniae, etc.

Fungal antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Dermatophytres, including: Epidermophyton floccusum,Microsporum audouini, Microsporum canis, Microsporum distortum,Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophytonconcentricum, Trichophyton equinum, Trichophyton gallinae, Trichophytongypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophytonquinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophytontonsurans, Trichophyton verrucosum, T. verrucosum var. album, var.discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophytonfaviforme; or from Aspergillus fumigatus, Aspergillus flavus,Aspergillus niger, Aspergillus nidulans, Aspergillus terreus,Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus,Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candidatropicalis, Candida glabrata, Candida krusei, Candida parapsilosis,Candida stellatoidea, Candida kusei, Candida parakwsei, Candidalusitaniae, Candida pseudotropicalis, Candida guilliermondi,Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis,Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum,Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp., Septataintestinalis and Enterocytozoon bieneusi; the less common are Brachiolaspp, Microsporidium spp., Nosema spp., Pleistophora spp.,Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis,Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale,Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe,Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii,Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaeaspp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolusspp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp,Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp,Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp,Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, andCladosporium spp.

Protazoan antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Entamoeba histolytica, Giardia lambli, Cryptosporidiumparvum, Cyclospora cayatanensis and Toxoplasma.

Plant antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Ricinus communis.

Suitable antigens include proteins and peptides from a virus such as,for example, human immunodeficiency virus (HIV), hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplexvirus (HSV), cytomegalovirus (CMV), influenza virus (flu), respiratorysyncytial virus (RSV), parvovorus, norovirus, human papilloma virus(HPV), rhinovirus, yellow fever virus, rabies virus, Dengue fever virus,measles virus, mumps virus, rubella virus, varicella zoster virus,enterovirus (e.g., enterovirus 71), ebola virus, and bovine diarrheavirus. Preferably, the antigenic substance is selected from the groupconsisting of HSV glycoprotein gD, HIV glycoprotein gp120, HIVglycoprotein gp 40, HIV p55 gag, and polypeptides from the pol and tatregions. In other preferred embodiments of the invention, the antigen isa protein or peptide derived from a bacterium such as, for example,Helicobacter pylori, Haemophilus influenza, Vibrio cholerae (cholera),C. diphtheriae (diphtheria), C. tetani (tetanus), Neisseriameningitidis, B. pertussis, Mycobacterium tuberculosis, and the like.

HIV antigens that can be encoded by the self-replicating RNA moleculesof the invention are described in U.S. application Ser. No. 490,858,filed Mar. 9, 1990, and published European application number 181150(May 14, 1986), as well as U.S. application Ser. Nos. 60/168,471;09/475,515; 09/475,504; and 09/610,313, the disclosures of which areincorporated herein by reference in their entirety.

Cytomegalovirus antigens that can be encoded by the self-replicating RNAmolecules of the invention are described in U.S. Pat. No. 4,689,225,U.S. application Ser. No. 367,363, filed Jun. 16, 1989 and PCTPublication WO 89/07143, the disclosures of which are incorporatedherein by reference in their entirety.

Hepatitis C antigens that can be encoded by the self-replicating RNAmolecules of the invention are described in PCT/US88/04125, publishedEuropean application number 318216 (May 31, 1989), published Japaneseapplication number 1-500565 filed Nov. 18, 1988, Canadian application583,561, and EPO 388,232, disclosures of which are incorporated hereinby reference in their entirety. A different set of HCV antigens isdescribed in European patent application 90/302866.0, filed Mar. 16,1990, and U.S. application Ser. No. 456,637, filed Dec. 21, 1989, andPCT/US90/01348, the disclosures of which are incorporated herein byreference in their entirety.

In some embodiments, the antigen is derived from an allergen, such aspollen allergens (tree-, herb, weed-, and grass pollen allergens);insect or arachnid allergens (inhalant, saliva and venom allergens, e.g.mite allergens, cockroach and midges allergens, hymenopthera venomallergens); animal hair and dandruff allergens (from e.g. dog, cat,horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Importantpollen allergens from trees, grasses and herbs are such originating fromthe taxonomic orders of Fagales, Oleales, Pinales and platanaceaeincluding, but not limited to, birch (Betula), alder (Alnus), hazel(Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria andJuniperus), plane tree (Platanus), the order of Poales including grassesof the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris,Secale, and Sorghum, the orders of Asterales and Urticales includingherbs of the genera Ambrosia, Artemisia, and Parietaria. Other importantinhalation allergens are those from house dust mites of the genusDermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and thosefrom mammals such as cat, dog and horse, venom allergens including suchoriginating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (Apidae), wasps(Vespidea), and ants (Formicoidae).

In certain embodiments, a tumor immunogen or antigen, or cancerimmunogen or antigen, can be encoded by the self-replicating RNAmolecule. In certain embodiments, the tumor immunogens and antigens arepeptide-containing tumor antigens, such as a polypeptide tumor antigenor glycoprotein tumor antigens.

Tumor immunogens and antigens appropriate for the use herein encompass awide variety of molecules, such as (a) polypeptide-containing tumorantigens, including polypeptides (which can range, for example, from8-20 amino acids in length, although lengths outside this range are alsocommon), lipopolypeptides and glycoproteins.

In certain embodiments, tumor immunogens are, for example, (a) fulllength molecules associated with cancer cells, (b) homologs and modifiedforms of the same, including molecules with deleted, added and/orsubstituted portions, and (c) fragments of the same. Tumor immunogensinclude, for example, class I-restricted antigens recognized by CD8+lymphocytes or class II-restricted antigens recognized by CD4+lymphocytes.

In certain embodiments, tumor immunogens include, but are not limitedto, (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well asRAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1,GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12(which can be used, for example, to address melanoma, lung, head andneck, NSCLC, breast, gastrointestinal, and bladder tumors), (b) mutatedantigens, for example, p53 (associated with various solid tumors, e.g.,colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g.,melanoma, pancreatic cancer and colorectal cancer), CDK4 (associatedwith, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8(associated with, e.g., head and neck cancer), CIA 0205 (associatedwith, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associatedwith, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkinslymphoma), BCR-abl (associated with, e.g., chronic myelogenousleukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT,(c) over-expressed antigens, for example, Galectin 4 (associated with,e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), WT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung and ovariancancer), alpha-fetoprotein (associated with, e.g., hepatoma), KSA(associated with, e.g., colorectal cancer), gastrin (associated with,e.g., pancreatic and gastric cancer), telomerase catalytic protein,MUC-1 (associated with, e.g., breast and ovarian cancer), G-250(associated with, e.g., renal cell carcinoma), p53 (associated with,e.g., breast, colon cancer), and carcinoembryonic antigen (associatedwith, e.g., breast cancer, lung cancer, and cancers of thegastrointestinal tract such as colorectal cancer), (d) shared antigens,for example, melanoma-melanocyte differentiation antigens such asMART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma), (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH—P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer, (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example).

In certain embodiments, tumor immunogens include, but are not limitedto, p15, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EpsteinBarr virus antigens, EBNA, human papillomavirus (HPV) antigens,including E6 and E7, hepatitis B and C virus antigens, human T-celllymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met,mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE,PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029,FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

C. Aqueous Solution for the Negatively Charged Molecule

The negatively charged molecule (such as RNA) is generally provided inthe form of an aqueous solution, or a form that can be readily dissolvedin an aqueous solution (e.g., lyophilized). The aqueous solution can bewater, or an aqueous solution that comprises a salt (e.g., NaCl), abuffer (e.g., a citrate buffer), an osmolality or tonicity adjustingagent (e.g., a saccharide), a polymer, a surfactant, or a combinationthereof. If the formulation is intended for in vivo administration, itis preferable that the aqueous solution is a physiologically acceptablebuffer that maintains a pH that is compatible with normal physiologicalconditions. Also, in certain instances, it may be desirable to maintainthe pH at a particular level in order to insure the stability of certaincomponents of the formulation.

For example, it may be desirable to prepare an aqueous solution that isisotonic and/or isosmotic. Hypertonic and hypotonic solutions sometimescould cause complications and undesirable effects when injected, such aspost-administration swelling or rapid absorption of the compositionbecause of differential ion concentrations between the composition andphysiological fluids. To control tonicity, the emulsion may comprise aphysiological salt, such as a sodium salt. Sodium chloride (NaCl), forexample, may be used at about 0.9% (w/v) (physiological saline). Othersalts that may be present include potassium chloride, potassiumdihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride,calcium chloride, etc. In an exemplary embodiment, the aqueous solutioncomprises 10 mM NaCl and other salts or non-ionic tonicifying agents. Asdescribed herein, non-ionic tonicifying agents can also be used tocontrol tonicity.

The aqueous solution may be buffered. Any physiologically acceptablebuffer may be used herein, such as citrate buffers, phosphate buffers,acetate buffers, succinate buffer, tris buffers, bicarbonate buffers,carbonate buffers, or the like. The pH of the aqueous solution willpreferably be between 6.0-8.0, preferably about 6.2 to about 6.8. Insome cases, certain amount of salt may be included in the buffer. Inother cases, salt in the buffer might interfere with complexation ofnegatively charged molecule to the emulsion particle, therefore isavoided.

The aqueous solution may also comprise additional components such asmolecules that change the osmolarity of the aqueous solution ormolecules that stabilizes the negatively charged molecule aftercomplexation. For example, the osmolality can be adjusted using anon-ionic tonicifying agent, which are generally carbohydrates but canalso be polymers. (See, e.g., Voet and Voet (1990) Biochemistry (JohnWiley & Sons, New York.) Examples of suitable non-ionic tonicifyingagents include sugars (e.g., trehalose, sucrose, dextrose, fructose,reduced palatinose, etc.), sugar alcohols (such as mannitol, sorbitol,xylitol, erythritol, lactitol, maltitol, glycerol, etc.), andcombinations thereof. If desired, a nonionic polymer (e.g., a poly(alkylglycol) such as polyethylene glycol, polypropylene glycol, orpolybutlyene glycol) or nonionic surfactant can be used. These types ofagents, in particular sugar and sugar alcohols, are also cryoprotectantsthat can procted RNA, and other negatibely charged molecules, whenlyophilized. In exemplary embodiments, the buffer comprises from about560 nM to 600 mM of trehalose, sucrose, sorbitol, or dextrose.

In some case, it may be preferable to prepare an aqueous solutioncomprising the negatively charged molecule as a hypertonic solution, andto prepare the cationic emulsion using unadulterated water or ahypotonic buffer. When the emulsion and the negatively charged moleculeare combined, the mixture becomes isotonic. For example, an aqueoussolution comprising RNA can be a 2× hypertonic solution, and thecationic emulsion can be prepared using 10 mM Citrate buffer. When theRNA solution and the emulsion are mixed at 1:1 (v/v) ratio, thecomposition becomes isotonic. Based on desired relative amounts of theemulsion to the aqueous solution that comprises the negatively chargedmolecule (e.g., 1:1 (v/v) mix, 2:1 (v/v) mix, 1:2 (v/v) mix, etc.), onecan readily determine the tonicity of the aqueous solution that isrequired in order to achieve an isotonic mixture.

Similarly, compositions that have physiological osmolality may bedesirable for in vivo administration. Physiological osmolality is fromabout 255 mOsm/kg water to about 315 mOsm/kg water. Sometimes, it may bepreferable to prepare an aqueous solution comprising the negativelycharged molecule as a hyperosmolar solution, and to prepare the cationicemulsion using unadulterated water or a hypoosmolar buffer. When theemulsion and the negatively charged molecule are combined, physiologicalosmolality is achieved. Based on desired relative amounts of theemulsion to the aqueous solution that comprises the negatively chargedmolecule (e.g., 1:1 (v/v) mix, 2:1 (v/v) mix, 1:2 (v/v) mix, etc.), onecan readily determine the osmolality of the aqueous solution that isrequired in order to achieve an iso-osmolar mixture.

In certain embodiments, the aqueous solution comprising the negativelycharged molecule may further comprise a polymer or a surfactant, or acombination thereof. In an exemplary embodiment, the oil-in-wateremulsion contains a poloxamer. In particular, the inventors haveobserved that adding Pluronic® F127 to the RNA aqueous solution prior tocomplexation to cationic emulsion particles led to greater stability andincreased RNase resistance of the RNA molecule. Addition of pluronicF127 to RNA aqueous solution was also found to decrease the particlesize of the RNA/CNE complex. Poloxamer polymers may also facilitateappropriate decomplexation/release of the RNA molecule, preventaggregation of the emulsion particles, and have immune modulatoryeffect. Other polymers that may be used include, e.g., Pluronic® F68 orPEG300.

Alternatively or in addition, the aqueous solution comprising thenegatively charged molecule may comprise from about 0.05% to about 20%(w/v) polymer. For example, the cationic oil-in-water emulsion maycomprise a polymer (e.g., a poloxamer such as Pluronic® F127, Pluronic®F68, or PEG300) at from about 0.05% to about 10% (w/v), such as 0.05%,0.05%_(,) 1%, or 5%_(.)

The buffer system may comprise any combination of two or more moleculesdescribed above (salt, buffer, saccharide, polymer, etc). In anpreferred embodiment, the buffer comprises 560 mM sucrose, 20 mM NaCl,and 2 mM Citrate, which can be mixed with a cationic oil in wateremulsion described herein to produce a final aqueous phase thatcomprises 280 mM sucrose, 10 mM NaCl and 1 mM citrate.

5. Methods of Preparation

In another aspect, the invention provides a method of preparing acomposition that comprises a negatively charged molecule complexed witha particle of a cationic oil-in-water emulsion, comprising: preparing acationic oil-in-water emulsion wherein the emulsion comprises: (1) fromabout 0.2% to about 20% (v/v) oil, (2) from about 0.01% to about 2.5%(v/v) surfactant, and (3) a cationic lipid; and adding the negativelycharged molecule to the cationic oil-in-water emulsion so that thenegatively charged molecule complexes with the particle of the emulsion.

One exemplary approach to generate the cationic oil-in-water emulsion isby a process comprising: (1) combining the oil and the cationic lipid toform the oil phase of the emulsion; (2) providing an aqueous solution toform the aqueous phase of the emulsion; and (3) dispersing the oil phasein the aqueous phase, for example, by homogenization. Homogenization maybe achieved in any suitable way, for example, using a commercialhomogenizer (e.g., IKA T25 homogenizer, available at VWR International(West Chester, Pa.).

The cationic lipid may be dissolved in a suitable solvent, such aschloroform (CHCl₃), dichloromethane (DCM), ethanol, acetone,Tetrahydrofuran (THF), 2,2,2 trifluoroethanol, acetonitrile, ethylacetate, hexane, Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO),etc., and added directly to the oil component of the emulsion.Alternatively, the cationic lipid may be added to a suitable solvent toform a liposome suspension; then the liposome suspension may be added tothe oil component of the emulsion. The cationic lipid may also bedissolved directly in the oil.

It may be desirable to heat the oil to a temperature between about 30°C. to about 65° C. to facilitate the dissolving of the lipid.

Desired amount of the cationic lipid (e.g., DOTAP) can be measured andeither dissolved in a solvent, in water, or directly in oil to reach adesired final concentration as described and exemplified herein.

Solvents such as chloroform (CHCl₃) or dichloromethane (DCM) may beremoved from the oil phase, e.g., by evaporation, prior to combining theaqueous phase and the oil phase or prior to homogenization.Alternatively, in instances where lipid solubility can be an issue, aprimary emulsion can be made with the solvent (e.g. DCM) still in theoil phase. In such cases, the solvent can be removed (e.g., allowed toevaporate) from the primary emulsion prior to a secondaryhomogenization.

If the emulsion comprises one or more surfactants, the surfactant(s) maybe included in the oil phase or the aqueous phase according to theconventional practice in the art. For example, SPAN85 can be dissolvedin the oil phase (e.g., squalene), and Tween 80 may be dissolved in theaqueous phase (e.g., in a citrate buffer).

In another aspect, the invention provides a method of preparing acomposition that comprises a negatively charged molecule (such as RNA)complexed with a particle of a cationic oil-in-water emulsion,comprising: (i) providing a cationic oil-in-water emulsion as describedherein; (ii) providing a aqueous solution comprising the negativelycharged molecule (such as RNA); and (iii) combining the oil-in-wateremulsion of (i) and the aqueous solution of (iii), so that thenegatively charged molecule complexes with the particle of the emulsion.For example, a cationic oil-in-water emulsion may be combined with anaqueous solution comprising a negatively charged molecule (e.g., anaqueous RNA solution) in any desired relative amounts, e.g., about 1:1(v/v), about 1.5:1 (v/v), about 2:1 (v/v), about 2.5:1 (v/v), about 3:1(v/v), about 3.5:1 (v/v), about 4:1 (v/v), about 5:1 (v/v), about 10:1(v/v), about 1:1.5 (v/v), about 1:2 (v/v), about 1:2.5 (v/v), about 1:3(v/v), about 1:3.5 (v/v), about 1:4 (v/v), about 1:1.5 (v/v), or about1:1.10 (v/v), etc.

The concentration of each component of the post-complex composition(e.g., RNA-emulsion complex) can be readily determined according torelative amounts of the pre-complex oil-in-water emulsion and theaqueous solution comprising the negatively charged molecule (e.g., anaqueous RNA solution) that are used. For example, when a cationicoil-in-water emulsion is combined with an aqueous solution comprising anegatively charged molecule (e.g., an aqueous RNA solution) at 1:1 (v:v)ratio, the concentrations of the oil and cationic lipid become ½ of thatof the pre-complex emulsion. Therefore, if an emulsion comprising 4.3%(w/v) squalene, 1.4 mg/mL DOTAP, 0.5% v/v SPAN85 and 0.5% v/v Tween 80(referred herein as “CNE17”) is combined with an aqueous RNA solutionthat comprises 560 mM sucrose, 20 mM NaCl, 2 mM Citrate, and 1% (w/v)Pluronic F127 at 1:1 (v:v), the post-complex composition comprises 2.15%(w/v) squalene, 0.7 mg/mL DOTAP, 0.25% v/v SPAN85, 0.25% v/v Tween 80,280 mM sucrose, 10 mM NaCl, 1 mM Citrate, and 0.5% (w/v) Pluronic F127.

Additional optional steps to promote particle formation, to improve thecomplexation between the negatively charged molecules and the cationicparticles, to increase the stability of the negatively charged molecule(e.g., to prevent degradation of an RNA molecule), to facilitateappropriate decomplexation/release of the negatively charged molecules(such as an RNA molecule), or to prevent aggregation of the emulsionparticles may be included. For example, a polymer (e.g., Pluronic® F127)or a surfactant may be added to the aqueous solution that comprises thenegatively charged molecule (such as RNA). In one exemplary embodiment,Pluronic® F127 is added to the RNA molecule prior to complexation to theemulsion particle.

The size of the emulsion particles can be varied by changing the ratioof surfactant to oil (increasing the ratio decreases droplet size),operating pressure (increasing operating pressure reduces droplet size),temperature (increasing temperature decreases droplet size), and otherprocess parameters. Actual particle size will also vary with theparticular surfactant, oil, and cationic lipid used, and with theparticular operating conditions selected. Emulsion particle size can beverified by use of sizing instruments, such as the commercial Sub-MicronParticle Analyzer (Model N4MD) manufactured by the Coulter Corporation,and the parameters can be varied using the guidelines set forth aboveuntil the average diameter of the particles is less than 1 μm, less than0.9 μm, less than 0.8 μm, less than 0.7 μm, less than 0.6 μm, less than0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, or lessthan 0.1 μm. Preferably, the particles have an average diameter of about400 nm or less, about 300 nm or less, about 200 nm or less, about 180 nmor less, about 150 nm or less, or about 140 nm or less, from about 50 nmto 180 nm, from about 60 nm to 180 nm, from about 70 to 180 nm, or fromabout 80 nm to 180 nm, from about 80 nm to 170 nm, from about 80 nm to160 nm, from about 80 nm to 150 nm, or from about 80 nm to 140 nm. Insome cases, it may be desirable that the mean particle size of thecationic emulsions is to 200 nm or less to allow for sterile filtration.In other cases, sterile filtration is not required and the mean particlesize of the cationic emulsions can be greater than 200 nm.

Optional processes for preparing the cationic oil-in-water emulsion(pre-complexation emulsion), or the negatively charged molecule-emulsioncomplex, include, e.g., sterilization, particle size selection (e.g.,removing large particles), filling, packaging, and labeling, etc.

For example, if the pre-complexation emulsion, or the negatively chargedmolecule-emulsion complex, is formulated for in vivo administration, itmay be sterilized, e.g., by filtering through a sterilizing grade filter(e.g., through a 0.22 micron filter). Other sterilization techniquesinclude a thermal process, or a radiation sterilization process, orusing pulsed light to produce a sterile composition.

The cationic oil-in-water emulsion described herein can be used tomanufacture vaccines. Sterile and/or clinical grade cationicoil-in-water emulsions can be prepared using similar methods asdescribed for MF59. See, e.g., Ott et al., Methods in MolecularMedicine, 2000, Volume 42, 211-228, in VACCINE ADJUVANTS (O'Hagan ed.),Humana Press. For example, similar to the manufacturing process of MF59,the oil phase and the aqueous phase of the emulsion can be combined andprocessed in an inline homogenizer to yield a coarse emulsion. Thecoarse emulsion can then be fed into a microfluidizer, where it can befurther processed to obtain a stable submicron emulsion. The coarseemulsion can be passed through the interaction chamber of themicrofluidizer repeatedly until the desired particle size is obtained.The bulk emulsion can then be filtered (e.g., though a 0.22-μm filterunder nitrogen) to remove large particles, yielding emulsion bulk thatcan be filled into suitable containers (e.g., glass bottles). Forvaccine antigens that have demonstrated long-term stability in thepresence of oil-in-water emulsion for self storage, the antigen andemulsion may be combined and sterile-filtered (e.g., though a 0.22-μmfilter membrane). The combined single vial vaccine can be filled intosingle-dose containers. For vaccine antigens where long-term stabilityhas not been demonstrated, the emulsion can be supplied as a separatevial. In such cases, the emulsion bulk can be filtered-sterilized (e.g.,though a 0.22-μm filter membrane), filled, and packaged in finalsingle-dose vials.

Quality control may be optionally performed on a small sample of theemulsion bulk or admixed vaccine, and the bulk or admixed vaccine willbe packaged into doses only if the sample passes the quality controltest.

6. Pharmaceutical Compositions and Administration

In another aspect, the invention provides a pharmaceutical compositioncomprising a negatively charged molecule complexed with a particle of acationic oil-in-water emulsion, as described herein, and may furthercomprise one or more pharmaceutically acceptable carriers, diluents, orexcipients. In preferred embodiments, the pharmaceutical composition isan immunogenic composition, which can be used as a vaccine.

Alternatively, the compositions described herein may be used to delivera negatively charged molecule to cells. For example, nucleic acidmolecules (e.g., DNA or RNA) can be delivered to cells for a variety ofpurposes, such as to induce production of a desired gene product (e.g.,protein), to regulate expression of a gene, for gene therapy and thelike. The compositions described herein may also be used to deliver anucleic acid molecule (e.g., DNA or RNA) to cells for therapeuticpurposes, such as to treat a disease such as cancers or proliferativedisorders, metabolic diseases, cardiovascular diseases, infections,allergies, to induce an immune response and the like. For example,nucleic acid molecules may be delivered to cells to inhibit theexpression of a target gene. Such nucleic acid molecules include, e.g.,antisense oligonucleotides, double-stranded RNAs, such as smallinterfering RNAs and the like. Double-stranded RNA molecules, such assmall interfering RNAs, can trigger RNA interference, which specificallysilences the corresponding target gene (gene knock down). Antisenseoligonucleotides are single strands of DNA or RNA that are complementaryto a chosen sequence. Generally, antisense RNA can prevent proteintranslation of certain messenger RNA strands by binding to them.Antisense DNA can be used to target a specific, complementary (coding ornon-coding) RNA. Therefore, the cationic emulsions described herein areuseful for delivering antisense oligonucleotides or double-stranded RNAsfor treatment of, for example, cancer by inhibiting production of anoncology target.

The pharmaceutical compositions provided herein may be administeredsingly or in combination with one or more additional therapeutic agents.The method of administration include, but are not limited to, oraladministration, rectal administration, parenteral administration,subcutaneous administration, intravenous administration, intravitrealadministration, intramuscular administration, inhalation, intranasaladministration, topical administration, ophthalmic administration, orotic administration.

A therapeutically effective amount of the compositions described hereinwill vary depending on, among others, the disease indicated, theseverity of the disease, the age and relative health of the subject, thepotency of the compound administered, the mode of administration and thetreatment desired.

In other embodiments, the pharmaceutical compositions described hereincan be administered in combination with one or more additionaltherapeutic agents. The additional therapeutic agents may include, butare not limited to antibiotics or antibacterial agents, antiemeticagents, antifungal agents, anti-inflammatory agents, antiviral agents,immunomodulatory agents, cytokines, antidepressants, hormones,alkylating agents, antimetabolites, antitumour antibiotics, antimitoticagents, topoisomerase inhibitors, cytostatic agents, anti-invasionagents, antiangiogenic agents, inhibitors of growth factor functioninhibitors of viral replication, viral enzyme inhibitors, anticanceragents, α-interferons, β-interferons, ribavirin, hormones, and othertoll-like receptor modulators, immunoglobulins (Igs), and antibodiesmodulating Ig function (such as anti-IgE (omalizumab)).

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of infectious diseases including, but notlimited to, disease cased by the pathogens disclosed herein, includingviral diseases such as genital warts, common warts, plantar warts,rabies, respiratory syncytial virus (RSV), hepatitis B, hepatitis C,Dengue virus, yellow fever, herpes simplex virus (by way of exampleonly, HSV-I, HSV-II, CMV, or VZV), molluscum contagiosum, vaccinia,variola, lentivirus, human immunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis virus (hepatitis C virus, hepatitis B virus,hepatitis A virus), cytomegalovirus (CMV), varicella zoster virus (VZV),rhinovirus, enterovirus (e.g. EV71), adenovirus, coronavirus (e.g.,SARS), influenza, para-influenza, mumps virus, measles virus, rubellavirus, papovavirus, hepadnavirus, flavivirus, retrovirus, arenavirus (byway of example only, LCM, Junin virus, Machupo virus, Guanarito virusand Lassa Fever) and Filovirus (by way of example only, ebola virus ormarburg virus).

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of bacterial, fungal, and protozoal infectionsincluding, but not limited to, malaria, tuberculosis and mycobacteriumavium, leprosy; pneumocystis carnii, cryptosporidiosis, histoplasmosis,toxoplasmosis, trypanosome infection, leishmaniasis, infections causedby bacteria of the genus Escherichia, Enterobacter, Salmonella,Staphylococcus, Klebsiella, Proteus, Pseudomonas, Streptococcus, andChlamydia, and fungal infections such as candidiasis, aspergillosis,histoplasmosis, and cryptococcal meningitis.

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of respiratory diseases and/or disorders,dermatological disorders, ocular diseases and/or disorders,genitourinary diseases and/or disorders including, allograft rejection,auto-immune and allergic, cancer, or damaged or ageing skin such asscarring and wrinkles.

In another aspect, the invention provides a method for generating orpotentiating an immune response in a subject in need thereof, such as amammal, comprising administering an effective amount of a composition asdisclosed herein. The immune response is preferably protective andpreferably involves antibodies and/or cell-mediated immunity. The methodmay be used to induce a primary immune response and/or to boost animmune response.

In certain embodiments, the compositions disclosed herein may be used asa medicament, e.g., for use in raising or enhancing an immune responsein a subject in need thereof, such as a mammal.

In certain embodiments, the compositions disclosed herein may be used inthe manufacture of a medicament for generating or potentiating an immuneresponse in a subject in need thereof, such as a mammal.

The invention also provides a delivery device pre-filled with acomposition or a vaccine disclosed herein.

The mammal is preferably a human, but may be, e.g., a cow, a pig, achicken, a cat or a dog, as the pathogens covered herein may beproblematic across a wide range of species. Where the vaccine is forprophylactic use, the human is preferably a child (e.g., a toddler orinfant), a teenager, or an adult; where the vaccine is for therapeuticuse, the human is preferably a teenager or an adult. A vaccine intendedfor children may also be administered to adults, e.g., to assess safety,dosage, immunogenicity, etc.

One way of checking efficacy of therapeutic treatment involvesmonitoring pathogen infection after administration of the compositionsor vaccines disclosed herein. One way of checking efficacy ofprophylactic treatment involves monitoring immune responses,systemically (such as monitoring the level of IgG1 and IgG2a production)and/or mucosally (such as monitoring the level of IgA production),against the antigen. Typically, antigen-specific serum antibodyresponses are determined post-immunization but pre-challenge whereasantigen-specific mucosal antibody responses are determinedpost-immunization and post-challenge.

Another way of assessing the immunogenicity of the compositions orvaccines disclosed herein where the nucleic acid molecule (e.g., theRNA) encodes a protein antigen is to express the protein antigenrecombinantly for screening patient sera or mucosal secretions byimmunoblot and/or microarrays. A positive reaction between the proteinand the patient sample indicates that the patient has mounted an immuneresponse to the protein in question. This method may also be used toidentify immunodominant antigens and/or epitopes within proteinantigens.

The efficacy of the compositions can also be determined in vivo bychallenging appropriate animal models of the pathogen of interestinfection.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

The compositions disclosed herein that include one or more antigens orare used in conjunction with one or more antigens may be used to treatboth children and adults. Thus a human subject may be less than 1 yearold, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55years old. Preferred subjects for receiving the compositions are theelderly (e.g., >50 years old, >60 years old, and preferably >65 years),the young (e.g., <5 years old), hospitalized patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. The compositions are notsuitable solely for these groups, however, and may be used moregenerally in a population.

The compositions disclosed herein that include one or more antigens orare used in conjunction with one or more antigens may be administered topatients at substantially the same time as (e.g., during the samemedical consultation or visit to a healthcare professional orvaccination centre) other vaccines, e.g., at substantially the same timeas a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine,a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine, a DTP vaccine, a conjugated H. influenzaetype b vaccine, an inactivated poliovirus vaccine, a hepatitis B virusvaccine, a meningococcal conjugate vaccine (such as a tetravalent A CW135 Y vaccine), a respiratory syncytial virus vaccine, etc.

In certain embodiments, the compositions provided herein include oroptionally include one or more immunoregulatory agents such asadjuvants. Exemplary adjuvants include, but are not limited to, a TH1adjuvant and/or a TH2 adjuvant, further discussed below. In certainembodiments, the adjuvants used in the immunogenic compositions provideherein include, but are not limited to:

-   -   1. Mineral-Containing Compositions;    -   2. Oil Emulsions;    -   3. Saponin Formulations;    -   4. Virosomes and Virus-Like Particles;    -   5. Bacterial or Microbial Derivatives;    -   6. Bioadhesives and Mucoadhesives;    -   7. Liposomes;    -   8. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations;    -   9. Polyphosphazene (PCPP);    -   10. Muramyl Peptides;    -   11. Imidazoquinolone Compounds;    -   12. Thiosemicarbazone Compounds;    -   13. Tryptanthrin Compounds;    -   14. Human Immunomodulators;    -   15. Lipopeptides;    -   16. Benzonaphthyridines;    -   17. Microparticles    -   18. Immunostimulatory polynucleotide (such as RNA or DNA; e.g.,        CpG-containing oligonucleotides)

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Development of Cationic Oil-in-Water Emulsions

Three types of cationic nanoemulsions (CNEs) were developed for thedelivery of self replicating RNA. Type 1 emulsions are “MF59” likeemulsions. These emulsions were made from the same components of MF59with the exception that cationic lipids are added. Type 2 emulsions areemulsions that replace the Span 85 and Tween 80 in MF59 withphospholipids. Type 3 emulsions are hybrid emulsions that are stabilizedby either lipids or other surfactants and can have additional polymersor surfactants in the aqueous phase of the emulsion.

Three different lipids were used in the preparation of Type 1 emulsions:1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP),3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride(DC Cholesterol) and Dimethyldioctadecylammonium (Bromide Salt) (DDA).DOTAP was used in the preparation of Type 2 and Type 3 emulsions.

The term N/P ratio refers to the amount of nitrogen in the cationiclipid in relation to the amount of phosphates on the RNA. The nitrogenis the charge bearing element within the cationic lipids tested. Thephosphate can be found on the RNA backbone. An N/P charge ratio of 10/1indicates that there are 10 positively charged nitrogens from thecationic lipid present for each negatively charged phosphate on the RNA.

Type 1 CNEs:

The ratio of Tween 80, Span 85, squalene, and citrate buffer were notchanged for this class of emulsions. These emulsions were prepared atthe same concentrations as MF59. The total amount of cationic lipidgiven per dose remains constant regardless of the lipid concentration.For example a 10 μg dose of RNA delivered in an emulsion with 0.8 mg/mlDOTAP emulsion at an N/P ratio of 10/1 would require a 2× dilution.Hence the amount of squalene delivered would be ½ of what is normallyadministered during immunization with MF59. Alternatively a 10 μg doseof RNA delivered in an emulsion with 1.6 mg/ml DOTAP at an N/P ratio of10/1 would require a 4× dilution.

In this example, 17 different formulations of Type 1 emulsions wereprepared. The ranges of cationic lipids that were able to be made intoemulsions are listed below:

TABLE 1 DOTAP 0.8 mg/ml up to 1.6 mg/ml DC cholesterol 0.62 mg/ml up to2.46 mg/ml DDA 0.73 mg/ml up to 1.64 mg/ml

Formulations with DOTAP concentrations of 0.8 mg/ml up to 1.6 mg/ml allproduced stable emulsions. Formulations with DC cholesterolconcentrations of 0.62 mg/ml up to 2.46 mg/ml all produced stableemulsions. Formulations with DDA concentrations of 0.73 mg/ml up to 1.64mg/ml all produced stable emulsions.

Type 2 CNEs:

The percentage of squalene varied with Type 2 CNEs. Another differencefrom MF59 is that these emulsions were made in water not in citratebuffer. These emulsions were made with1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) andphosphatidylcholine (egg PC) as lipid stabilizers. Emulsions were madeusing DOPE and egg PC with either DOTAP, DC cholesterol or DDA at theoptimized concentrations from the type 1 emulsion studies.

A separate series of emulsions were made using only DOTAP as thestabilizer. These emulsions contained various amounts of squalene (from0.43% w/w up to the MF59 concentration of 4.3% w/w).

Type 3 CNEs:

The addition of Pluronic® F127 (poloxomer) to the RNA prior tocomplexation to a DOTAP/Egg PC emulsion led to greater RNase stabilitywhen compared to a sample that did not have the poloxamer added to it.This indicates the role of this polymer in allowing for better RNAcomplexation with the oil droplet.

The addition of a small amount of tween 80 (0.08% w/w) during theemulsification step of the DOTAP-only emulsions led to a smaller dropletsize.

Methods of Preparing Cationic Emulsions:

Squalene, sorbitan trioleate (Span 85), polyoxy-ethylene sorbitanmonololeate (Tween 80) were obtained from Sigma (St. Louis, Mo., USA).Dimethyldioctadecylammonium (DDA),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),3β-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl), were purchased from Avanti Lipids.L-α-lysophosphatidylcholine (Egg, Chicken) and1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased fromLipoid (Ludwigshafen Germany).

Cationic nanoemulsions (CNEs) were prepared similar to charged MF59 aspreviously described with minor modifications (Ott, Singh et al. 2002).Briefly, oil soluble components (ie. Squalene, span 85, cationic lipids,lipid surfactants) were combined in a beaker, lipid components weredissolved in chloroform (CHCl₃) or dichloromethane (DCM). The resultinglipid solution was added directly to the oil plus span 85. For a subsetof emulsions (CNE01, 02, 17) the solvent was allowed to evaporate atroom temperature for 2 hours in a fume hood prior to combining theaqueous phase and homogenizing the sample. For the remaining emulsions(CNE 12, 13, 27, 32, 35), the oil phase was combined with the aqueousphase and immediately homogenized for 2 min using an IKA T25 homogenizerat 24K RPM in order to provide a homogeneous feedstock. CNE05 wasprepared by preparing a liposome stock solution. Liposomes were preparedby evaporating the 10 mg/ml DOTAP chloroform solution using a rotaryevaporator (Buchi model number R200) at 300 milliTorr, pressure for 30minutes at a temperature of 50° C. Residual chloroform evaporation wasinsured by placing the samples overnight in a Labconco freeze dryer. Thelipid film was hydrated and dispersed by adding 1.0 mL of filtereddeionized distilled water and placed at 50° C. to ensure full suspensionof the lipid. The resulting liposomes were added directly to thesqualene and were immediately emulsified for 2 min using an IKA T25homogenizer at 24K RPM. Emulsions were then allowed to sit at roomtemperature on a stirplate for 2-3 hours after primary homogenization ina fume hood. The primary emulsions were passed three to five timesthrough a Microfluidezer M110S homogenizer with an ice bath cooling coilat a homogenization pressure of approximately 15 k-20 k PSI(Microfluidics, Newton, Mass.). The 20 ml batch samples were removedfrom the unit and stored at 4° C. Table 2 describes the components ofthe emulsions.

TABLE 2 mg/ml + CNE Cationic Lipid (+) Lipid Surfactant SqualeneBuffer/water CNE01 DOTAP 0.8 0.5% SPAN 85 4.3% 10 mM citrate (in CHCl3)0.5% Tween 80 buffer pH 6.5 CNE02 DOTAP 1.6 0.5% SPAN 85 4.3% 10 mMcitrate (in CHCl3) 0.5% Tween 80 buffer pH 6.5 CNE05 DOTAP 1.2 0.08%Tween 80 0.5% DEPC (in CHCl3) treated water CNE12 DC Cholesterol 2.460.5% SPAN 85 4.3% 10 mM citrate (in DCM) 0.5% Tween 80 buffer pH 6.5CNE13 DDA 1.45 0.5% SPAN 85 4.3% 10 mM citrate (in DCM) 0.5% Tween 80buffer pH 6.5 CNE17 DOTAP 1.40 0.5% SPAN 85 4.3% 10 mM citrate (in DCM)0.5% Tween 80 buffer pH 6.5 CNE27 DOTAP (in DCM) + 1.40 — 4.3%Rnase-free 30 mg DOPE dH₂O CNE32 DOTAP (in DCM) + 1.40 — 4.3% Rnase-free30.9 mg Egg PC dH₂O CNE35 DOTAP (in DCM) + 1.40 — 4.3% Rnase-free 32.16mg DPyPE dH₂O

One method of addition of the lipids into the oil phase of the emulsionswas adding dichloromethane (DCM or methylene chloride) into the oilphase. Once added the DCM could be allowed to evaporate fully. Afterevaporation, emulsion was then passed through the Microfluidizer.Alternatively, in instances where lipid solubility was an issue theprimary emulsion could be made with the DCM still in the organic phase.In that case, the DCM would be allowed to evaporate directly from theemulsion prior to secondary homogenization.

An alternative method for emulsions that contained lipids as stabilizerswas to make a lipid film and rehydrate the film, so that the lipidsformed liposomes. The liposomes were then added to the oil phase andprocessed as standard MF59 was processed.

Example 2 Preparing RNA-Particle Complexes 1. Materials and Methods. RNASynthesis

Plasmid DNA encoding an alphavirus replicon (self-replicating RNA) wasused as a template for synthesis of RNA in vitro. Each replicon containsthe genetic elements required for RNA replication but lacks sequencesencoding gene products that are necessary for particle assembly. Thestructural genes of the alphavirus genome were replaced by sequencesencoding a heterologous protein (whose expression is driven by thealphavirus subgenomic promoter). Upon delivery of the replicons toeukaryotic cells, the positive-stranded RNA is translated to producefour non-structural proteins, which together replicate the genomic RNAand transcribe abundant subgenomic mRNAs encoding the heterologousprotein. Due to the lack of expression of the alphavirus structuralproteins, replicons are incapable of generating infectious particles. Abacteriophage T7 promoter is located upstream of the alphavirus cDNA tofacilitate the synthesis of the replicon RNA in vitro, and the hepatitisdelta virus (HDV) ribozyme located immediately downstream of thepoly(A)-tail generates the correct 3′-end through its self-cleavingactivity. The sequences of the four plasmids used in the examples areshown in FIGS. 7A-7B.

Following linearization of the plasmid DNA downstream of the HDVribozyme with a suitable restriction endonuclease, run-off transcriptswere synthesized in vitro using T7 or SP6 bacteriophage derivedDNA-dependent RNA polymerase. Transcriptions were performed for 2 hoursat 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNApolymerase) final concentration of each of the nucleoside triphosphates(ATP, CTP, GTP and UTP) following the instructions provided by themanufacturer (Ambion, Austin, Tex.). Following transcription, thetemplate DNA was digested with TURBO DNase (Ambion, Austin, Tex.). Thereplicon RNA was precipitated with LiCl and reconstituted innuclease-free water. Uncapped RNA was capped post-transcriptionally withVaccinia Capping Enzyme (VCE) using the ScriptCap m⁷G Capping System(Epicentre Biotechnologies, Madison, Wis.) as outlined in the usermanual. Post-transcriptionally capped RNA was precipitated with LiCl andreconstituted in nuclease-free water. Alternatively, replicons may becapped by supplementing the transcription reactions with 6 mM (for T7RNA polymerase) or 4 mM (for SP6 RNA polymerase) m⁷G(5′)ppp(5′)G, anonreversible cap structure analog (New England Biolabs, Beverly, Mass.)and lowering the concentration of guanosine triphosphate to 1.5 mM (forT7 RNA polymerase) or 1 mM (for SP6 RNA polymerase). The transcripts maybe then purified by TURBO DNase (Ambion, Austin, Tex.) digestionfollowed by LiCL precipitation and a wash in 75% ethanol.

The concentration of the RNA samples was determined by measuring theoptical density at 260 nm. Integrity of the in vitro transcripts wasconfirmed by denaturing agarose gel electrophoresis for the presence ofthe full length construct.

RNA Complexation

The number of nitrogens in solution was calculated from the cationiclipid concentration, DOTAP for example has 1 nitrogen that can beprotonated per molecule. The RNA concentration was used to calculate theamount of phosphate in solution using an estimate of 3 nmols ofphosphate per microgram of RNA. By varying the amount of RNA:Lipid, theN/P ratio can be modified. RNA was complexed to the CNEs in a range ofnitrogen/phosphate ratios (N/P). Calculation of the N/P ratio was doneby calculating the number of moles of protonatable nitrogens in theemulsion per milliliter. To calculate the number of phosphates, aconstant of 3 nmols of phosphate per microgram of RNA was used. Afterthe values were determined, the appropriate ratio of the emulsion wasadded to the RNA. Using these values, the RNA was diluted to theappropriate concentration and added directly into an equal volume ofemulsion while vortexing lightly. The solution was allowed to sit atroom temperature for approximately 2 hours. Once complexed the resultingsolution was diluted to the appropriate concentration and used within 1hour.

Gel Electrophoresis

Denaturing gel electrophoresis was performed to assess binding of RNAwith the cationic formulations and stability in the presence of RNase A.The gel was cast as follows: 2 g of agarose (Bio-Rad, Hercules, Calif.)was added to 180 ml of water and heated in a microwave until dissolvedand then cooled to 60° C. 20 ml of 10× denaturing gel buffer (Ambion,Austin, Tex.), was then added to the agarose solution. The gel waspoured and was allowed to set for at least 45 minutes at roomtemperature. The gel was then placed in a gel tank, and 1×MOPS runningbuffer (Ambion, Austin, Tex.) was added to cover the gel by a fewmillimeters.

RNase Protection Assay

RNase digestion was achieved by incubation with 6.4 mAU of RNase A permicrogram of RNA (Ambion, Hercules, Calif.) for 30 minutes at roomtemperature. RNase was inactivated with Proteinase K (Novagen,Darmstadt, Germany) by incubating the sample at 55° C. for 10 minutes.Post-RNase inactivation samples were decomplexed with a 1:1 mixture ofsample to 25:24:1, phenol:chloroform:isoamyl alcohol. Samples wereinverted several times to mix and then placed on a centrifuge for 15minutes at 12 k RPM. The aqueous phase was removed from the organicphase and used to analyze the RNA. Prior to loading (460 ng per well)all the samples were incubated with formaldehyde loading dye, denaturedfor 10 minutes at 65° C. and cooled to room temperature. AmbionMillennium markers were used to approximate the molecular weight of theRNA construct. The gel was run at 130 V. The gel was stained using 0.1%SYBR gold according to the manufacturer's guidelines (Invitrogen,Carlsbad, Calif.) in water by rocking at room temperature for 1 hour.Gel images were taken on a Bio-Rad Chemidoc XRS imaging system(Hercules, Calif.). All studies used mouse thymus RNA from Clonetech(Mountain View, Calif.).

Heparin Binding Assay

RNA was complexed as described above. The RNA/CNE complex was incubatedwith various concentrations of heparin sulfate (Alfa Aesar, Ward HillMass.) for 30 minutes at room temperature. The resulting solutions werecentrifuged for 15-20 minutes minutes. The centrifuge tubes werepunctured with a tuberculin syringe and the subnatant was removed. Thesolution was then assayed for RNA concentration using the Quant-itRibogreen RNA Assay Kit (Invitrogen, Carlsbad Calif.) according to themanufacturer's directions. The samples were analyzed on a Biotek Synergy4 (Winooski, Vt.) fluorescent plate reader. Free RNA values werecalculated using a standard curve.

Particle Size Assay

Particle size of the emulsion was measured using a Zetasizer Nano ZS(Malvern Instruments, Worcestershire, UK) according to themanufacturer's instructions. Particle sizes are reported as theZ-Average (ZAve) with the polydispersity index (pdi). All samples werediluted in water prior to measurements. Additionally, particle size ofthe emulsion was measured using Horiba LA-930 particle sizer (HoribaScientific, USA). Samples were diluted in water prior to measurements.Zeta potential was measured using Zetasizer Nano ZS using dilutedsamples according to the manufacturer's instructions.

Secreted Alkaline Phosphatase (SEAP) Assay

To assess the kinetics and amount of antigen production, an RNA repliconencoding for SEAP was administered with and without formulation to miceintramuscularly. Groups of 3 or 5 female Balb/C mice aged 8-10 weeks andweighing about 20 g were immunized with CNEs complexed with replicon RNAencoding for SEAP at the indicated N/P ratios. Naked RNA was formulatedin RNase free 1×PBS. A 100 μl dose was administered to each mouse (50 μlper site) in the quadriceps muscle. Blood samples were taken 1, 3, and 6days post injection. Serum was separated from the blood immediatelyafter collection, and stored at −30° C. until use.

A chemiluminescent SEAP assay Phospha-Light System (Applied Biosystems,Bedford, Mass.) was used to analyze the serum. Mouse sera was diluted1:4 in 1× Phospha-Light dilution buffer. Samples were placed in a waterbath sealed with aluminum sealing foil and heat inactivated for 30minutes at 65° C. After cooling on ice for 3 minutes, and equilibratingto room temperature, 50 uL of Phospha-Light assay buffer was added tothe wells and the samples were left at room temperature for 5 minutes.Then, 50 uL of reaction buffer containing 1:20 CSPD® (chemiluminecentalkaline phosphate substrate) substrate was added, and the luminescencewas measured after 20 minutes of incubation at room temperature.Luminescence was measured on a Berthold Centro LB 960 luminometer (OakRidge, Tenn.) with a 1 second integration per well. The activity of SEAPin each sample was measured in duplicate and the mean of these twomeasurements is shown.

Electroporation

Electroporation was a very effective method for the delivery of plasmidDNA vaccines and this technique was used to delivery self-replicatingRNA. Mice were anesthetized under isofluorane, both hind legs wereclosely shaven to expose the area on the limb to be treated. A dose of30 μl of vaccine was injected to the quadracepts muscle of the hind limbusing a ½ cc insulin syringe. The muscle was electroporated using theElgen® DNA Delivery System (Inovio, San Diego). The instrumentparameters are as follows: 60V, 2 pulses each at 60 ms. Another dose wassimilarly delivered to the second limb, followed by electroporation.

Viral Replicon Particles (VRP)

To compare RNA vaccines to traditional RNA-vectored approaches forachieving in vivo expression of reporter genes or antigens, we utilizedviral replicon particles (VRPs) produced in BHK cells by the methodsdescribed by Perri et al. In this system, the antigen (or reporter gene)replicons consisted of alphavirus chimeric replicons (VCR) derived fromthe genome of Venezuelan equine encephalitis virus (VEEV) engineered tocontain the 3′ terminal sequences (3′ UTR) of Sindbis virus and aSindbis virus packaging signal (PS) (see FIG. 2 of Perri S., et al., JVirol 77: 10394-10403 (2003)). These replicons were packaged into VRPsby co-electroporating them into baby hamster kidney (BHK) cells alongwith defective helper RNAs encoding the Sindbis virus capsid andglycoprotein genes (see FIG. 2 of Perri et al). The VRPs were thenharvested and titrated by standard methods and inoculated into animalsin culture fluid or other isotonic buffers.

2. Particle Size Analysis of the Oil-in-Water Emulsions.

After manufacture the emulsions were analyzed for particle size and zetapotential. Tables 3 and 4 summarize the data particle size and zetapotential data obtained pre and post complexation at an N/P ratio of 4:1and 10:1. Particle size of the emulsions was below 160 nm for all of theformulations tested when measured on the Nano ZS particle sizer. Aftercomplexation some of the particle sizes did increase significantlyparticularly at the 4:1 N/P ratio. This is likely due to the aggregationand bridging of the RNA between multiple emulsion droplets. Horriba datagenerally matched well with the NanoZS measurements except for a fewcases (CNE02 and CNE32) where there seem to be a larger particlepopulation that is not able to be analyzed on the nanoZS. All CNEs withthe exception of CNE02 and CNE32 were less than 190 nm in size whenmeasured on the Horiba particle sizer. The low variability in sizeindicates a robust processing method regardless of the amount or type ofcationic lipid added. It is particularly desirable that the meanparticle size to be below 200 nm in size to allow for sterilefiltration. All samples tested pass this criterion.

TABLE 3 Particle size data Horiba Measurement Nano ZS measurement NotNot Formulation complexed complexed 4:1 N/P 10:1 N/P CNE01 187.8 159.6156.7 141.9 CNE02 535 121.9 — — CNE05 — 110.1 143.6 132.3 CNE12 127.1124 366.6 153 CNE13 128.5 117.4 273.3 163.4 CNE17 129.4 134.8 — 164CNE27 137.2 134.6 223.5 139.2 CNE32 279.8 114.8 185.4 134.7 CNE35 —134.7 161.1 142.7

Zeta potential was slightly more variable than the particle size data(Table 4). This is in line with our expectations since a number of thedifferences in these formulations is the change in cationic lipidconcentration. For example CNE01, CNE02, and CNE17 each contain 0.8, 1.6and 1.2 mg/ml of DOTAP respectively. The zeta potential for these lotswere in line with expectations with CNE01 having the lowest zetapotential of 15.9 mV pre complexation, followed by CNE17 with apre-complexation zeta potential of 33.4 mV, and lastly CNE02 with a zetapotential of 43 mV. The zeta potential post complexation is generallynot changing much from the pre-complexation zeta potential likely due tothe excess charge in the emulsions.

TABLE 4 Zeta potential Not complexed 4:1 N/P 10:1 N/P Formulation (mV)(mV) (mV) CNE01 15.9 41.4 36.6 CNE02 43 — — CNE05 74.2 44.9 15.2 CNE1224.5 18.2 24.8 CNE13 26.3 33.2 33.4 CNE17 33.4 33.9 30.7 CNE27 63.2 25.126.8 CNE32 66.9 39.2 27.6 CNE35 78 23.9 43.6

3. RNase stability assay:

To assess the ability of the emulsions to protect from RNase degradationan in-vitro assay was developed to screen formulations. FIG. 1 shows theresults of RNase protection assay of CNE01 and CNE17 at a 10:1 and 4:1N/P ratio. CNE01 protected the RNA better at a 10:1 ratio compared tothe 4:1 ratio. CNE17 showed good protection at 10:1.

FIG. 2 shows that CNE17 was also able to protect the RNA at an N/P ratioof 4:1. CNE12 and 13 also protected the RNA (similar to CNE17) at bothcharge ratios (FIG. 2). FIG. 3 shows similar results as FIG. 1 withCNE01 not protecting very well at a 4:1 N/P ratio. CNE02 did protectagainst RNases very well at both N/P ratios tested (FIGS. 3 and 4).CNE04 did not protect the RNA from RNase digestion, but CNE05 was ableto protect the RNA at both charge ratios tested (FIG. 5). CNE27 showedvery little RNase protection, while CNE32 showed slightly moreprotection, but overall less than the previously mentioned formulations.CNE 35 (FIG. 6) was able to slightly protect the RNA from degradationfrom RNase. Overall 5 different formulations were able to preventdegradation of the RNA in vitro.

4. In Vivo SEAP Screening:

A306 replicon, which expresses secreted alkaline phosphatase (SEAP), wasused to determine the protein expression level in vivo afteradministration of alphavirus vectors. BALB/c mice, 5 animals per group,were given bilateral intramuscular vaccinations (50 μL per leg) on days0 with VRP's expressing SEAP (5×10⁵ IU), naked self-replicating RNA(A306, 1 μg), self-replicating RNA delivered using electroporation(A306+EP, 1 and 0.1 μg, respectively) and self-replicating RNAformulated with CNE17, CNE05 and CNE35 at an N/P ratio of 10:1 producedas previously described (1 μg or 0.1 g A306,). Serum SEAP levels(relative light units, RLU) on days 1, 3 and 6 after intramuscularvaccination on day 0 are shown in Table 5. Data are represented asarithmetic mean titers of 5 individual mice per group.

TABLE 5 Group Dose (μg) DAY1 DAY3 DAY6 VRP 5 × 10{circumflex over ( )}5IU 161,428 46,594 35,998 A306 1 2,992 35,000 228,614 CNE17 1 4,61554,108 570,484 CNE17 0.1 2,509 14,772 157,386 A306 + EP 1 2,047 18,208173,176 A306 + EP 0.1 1,745 8,249 56,927 CNE05 1 1,831 1,748 5,171 CNE351 1,712 1,811 11,005

Table 5 shows that serum SEAP levels increased when the RNA wasformulated in CNE17 relative to the naked RNA control at a similar dose.SEAP expression was increase when the RNA was formulated in the CNErelative to the VRP control, but the kinetics of expression was verydifferent. Delivery with electroporation resulted in increased SEAPexpression relative to the naked RNA control, but these levels werelower as compared to the SEAP expression level when the RNA wasformulated with CNE17. CNE05 and CNE35 reduced protein expression level.

5. Effect of N/P Ratios on SEAP Expression (CNE17)

A306 replicon, which expresses secreted alkaline phosphatase (SEAP), wasused to determine the protein expression level in vivo afteradministration of alphavirus vectors. BALB/c mice, 5 animals per group,were given bilateral intramuscular vaccinations (50 μL per leg) on days0 with naked self-replicating RNA (A306, 1 μg), self-replicating RNAformulated with CNE17, produced as previously described (A306, 1 ng) atthe following N/P ratio's 6:1, 7:1, 8:1, 10:1, 12:1, 13:1, 14:1, 16:1.

Serum SEAP levels (relative light units, RLU) on days 1, 3 and 6 afterintramuscular vaccination on day 0 are shown in Table 6. Data arerepresented as arithmetic mean titers of 5 individual mice per group. Acorrelation of the heparin sulfate binding compared to day 6 SEAPexpression is outlined in Table 7. Percentages of RNA released from thecomplex at 6×, 8×, and 10× heparin sulfate, respectively, are indicated.

TABLE 6 Serum SEAP levels (CNE17) A306 Dose Group (μg) DAY1 DAY3 DAY6A306 1 1,235 3,271 5,215 CNE17 6:1 1 6,189 17,960 131,321 CNE17 7:1 12,836 40,736 266,217 CNE17 8:1 1 5,737 26,823 316,274 CNE17 10:1 1 8,01831,988 333,184 CNE17 12:1 1 7,775 23,412 295,218 CNE17 13:1 1 9,21724,236 247,262 CNE17 14:1 1 7,317 26,072 279,585 CNE17 16:1 1 15,01417,141 144,582

TABLE 7 Heparin binding and day 6 SEAP expression (CNE17) N/P 6x heparin8x heparin 10x heparin Day 6 SEAP Standard ratio Sulfate Sulfate Sulfateexpression Deviation 4 5.4  4.9 4.6 — — 6 7.7 16.6 32.1 131321  49229 715.2 27.1 42.8 266217 190144 8 20.7 39.8 50.8 316274 138669 10 53.9 72.779.3 333184 168456 12 45.4 71.7 88.8 295218 153891 13 — — — 247262 85926 14 13.1 84.1 81.5 279585 261205 16 1 47.5 84.9 144583 105973 18 021.1 78 — —

Tables 6 and 7 show that serum SEAP levels increased when the RNA wasformulated at an N/P ratio of 10:1 relative to the naked RNA control ata similar dose. The other N/P ratio's tested expressed lower amounts ofprotein expression as compared to the 10:1 N/P ratio, but all showed ahigher response than naked RNA. It should be highlighted that theaverage SEAP values from the naked RNA fluctuated considerably, which isexemplified in Tables 5 and 6, with expression of at approximately35,000 in one experiment, and 5,000 in another. The protein expressionlevel on day 6 correlated well with the heparin release.

6. Effect of N/P Ratios on SEAP Expression (CNE13)

A306 replicon, which expresses secreted alkaline phosphatase (SEAP), wasused to determine the protein expression level in-vivo afteradministration of alphavirus vectors. BALB/c mice, 5 animals per group,were given bilateral intramuscular vaccinations (50 μL per leg) on days0 with naked self-replicating RNA (A306, 1 μg), self-replicating RNAformulated with CNE13, produced as previously described (1 μg A306) atthe following N/P ratio's 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1.

Serum SEAP levels (relative light units, RLU) on days 1, 3 and 6 afterintramuscular vaccination on day 0 are shown in Table 8. Data arerepresented as arithmetic mean titers of 5 individual mice per group. Acorrelation of the heparin sulfate binding compared to day 6 SEAPexpression is outlined in Table 9. Percentages of RNA released from thecomplex at 6×, 8×, and 10× heparin sulfate, respectively, are indicated.

TABLE 8 Serum SEAP levels (CNE13) A306 Dose Group (μg) DAY1 DAY3 DAY6A306 1 1,507 42,405 138,978 CNE13 18:1 1 5,425 169,971 1,104,679 CNE1316:1 1 3,584 68,118 586,874 CNE13 14:1 1 5,199 56,314 745,815 CNE13 12:11 3,609 212,772 1,462,864 CNE13 10:1 1 5,538 200,506 1,103,004 CNE13 8:11 6,038 95,870 872,715 CNE13 6:1 1 4,116 23,000 291,485

TABLE 9 Heparin binding and day 6 SEAP expression (CNE13) N/P 6x heparin8x heparin 10x heparin Day 6 SEAP Standard ratio Sulfate Sulfate Sulfateexpression Deviation 4 6.94 7.81 8.6 — — 6 10.9 13.02 14.48 291485313966 8 19.33 24.44 29.01 872715 530829 10 27.64 33.57 39.1 11030041095207 12 22.85 40.28 45.95 1462864 1413440 14 19.3 35.91 40.97 745815415278 16 6.23 34.86 42.45 586875 471111 18 0.71 28.32 40.47 1104680715503 20 0.32 13.77 42.64 — —

Tables 8 and 9 show that serum SEAP levels increased when the RNA wasformulated at all N/P ratio's tested relative to the naked RNA controlat similar dose. The protein expression on day 6 correlated well withthe heparin release.

7. Effect of N/P Ratios on SEAP Expression (CNE01)

A306 replicon, which expresses secreted alkaline phosphatase (SEAP), wasused to determine the protein expression level in-vivo afteradministration of alphavirus vectors. BALB/c mice, 5 animals per group,were given bilateral intramuscular vaccinations (50 μL per leg) on day 0with naked self-replicating RNA (A306, 1 μg), self-replicating RNAformulated with CNE01, produced as previously described (1 μg A306) atthe following N/P ratios 4:1, 10:1, 12:1, 14:1, 16:1, 18:1.

Serum SEAP levels (relative light units, RLU) on days 1, 3 and 6 afterintramuscular vaccination on day 0 are shown in Table 10. Data arerepresented as arithmetic mean relative light units (RLUs) of 5individual mice per group. A correlation of the heparin sulfate bindingcompared to day 6 SEAP expression is outlined in Table 11. Percentagesof RNA released from the complex at 6×, 8×, and 10× heparin sulfate,respectively, are indicated.

TABLE 10 Serum SEAP levels (CNE01) A306 Dose Group (μg) DAY1 DAY3 DAY6A306 1 9,102 6,567 17,994 CNE01 4:1 1 4,326 8,064 104,097 CNE01 10:1 15,865 14,058 237,271 CNE01 12:1 1 19,365 14,096 117,644 CNE01 14:1 14,841 11,531 148,937 CNE01 16:1 1 9,061 20,639 182,854 CNE01 18:1 113,822 45,073 285,868

TABLE 11 Heparin binding and day 6 SEAP expression of CNE01 N/P 6xheparin 8x heparin 10x heparin Day 6 SEAP Standard ratio Sulfate SulfateSulfate expression Deviation 4 59.76 66.5 69.18 104098 64504 6 70.6673.36 72.05 — — 8 69.96 71.36 69.14 — — 10 66.89 66.63 63.06 23727150946 12 58.91 55.05 51.42 117645 64871 14 55.57 45.65 38.91 14893828513 16 52.89 39 32.36 182854 36627 18 42.42 35.21 27.74 285868 8325119 30.04 40.73 27.63 — —

Tables 10 and 11 show that serum SEAP levels increased when the RNA wasformulated at all N/P ratio's tested relative to the naked RNA controlat similar dose.

Example 3 Assessing Protein Expression Levels Using Different Oils

A series of emulsions were made using different oils but within the baseformulation of CNE17, i.e., 5% oil, 0.5% Tween 80, 0.5% span 85 and 1.4mg/ml DOTAP. Table 12 below outlines the changes in oils for each of thegroups. Classifications of the oils are also listed in Table 12.

The emulsions were tested at a 10:1 N/P ratio and was complexed aspreviously described. BALB/c mice, 5 animals per group, were givenbilateral intramuscular vaccinations (50 μL per leg) on day 0 with VRP'sexpressing SEAP (5×10⁵ IU), naked self-replicating RNA (A306, 1 μg),self-replicating RNA delivered using electroporation (A306+EP, 1 and 0.1μg) and self-replicating RNA formulated with CNE36, CNE37, CNE38, andCNE41 produced as previously described (1 μg A306,). Serum SEAP levels(relative light units, RLU) on days 1, 3 and 6 after intramuscularvaccination on day 0 are shown in Table 13. Data are represented asarithmetic mean RLUs of 5 individual mice per group.

TABLE 12 Emulsion Type of oil Source and composition CNE17 SqualeneShark liver oil, triterpene CNE36 Soybean oil Non-animal derived oil,triglycerides made up of alpha- linolenic, linoleic, oleic, stearic andpalmitic acids. CNE37 Cod liver oil Fish liver oil, high levels ofomega-3 fatty acids (eicosapentaenoic acid, decosahexaenoic acid), vit Aand vitamin D. CNE38 Sunflower oil Non-animal derived oil, primarilylinoleic acid triglycerides (~50%), much lesser amounts of oleic,stearic and palmitic acid CNE41 Olive oil Non-animal derived oil,triglycerides of oleic, palmitic and other fatty acids

TABLE 13 Group Dose (μg) DAY1 DAY3 DAY6 A306 + EP 1,403 49,969 179,916CNE36 1,506 3,288 83,268 CNE37 1,387 1,127 1,594 CNE38 1,791 2,22847,705 CNE41 1,369 2,113 60,039 VRP 5 × 10{circumflex over ( )}5 IU105,829 38,546 56,155 A306 1,212 6,007 95,380 A306 + MF59 1,219 1,65611,667

As shown in Table 13, CNE17 shows the highest level of expressionthroughout the studies. All of the other emulsions were inferior to a 1μg dose of naked RNA. CNE36 resulted in highest expression of the newoils, followed by CNE41 and CNE38. A 1 μg dose of RNA added directly toMF59 muted the response.

Example 4 CNE17 Enhanced Immunogenicity of RSV-F Antigen in a MouseModel 1. Methods Murine Immunogenicity Studies

The A317 replicon that expresses the surface fusion glycoprotein of RSV(RSV-F) was used for this study. BALB/c mice, aged 8-10 weeks andweighing about 20 g, 10 animals per group, were given bilateralintramuscular vaccinations. All animals were injected in the quadricepsin the two hind legs each getting an equivalent volume (50 μL per leg)on days 0 and 21 with VRP's expressing RSV-F (1×10⁶ IU), nakedself-replicating RNA (A317, 1 μg), self-replicating RNA delivered usingelectroporation (10 μg A317+EP), or self-replicating RNA formulated inCNE17 (0.1 μg or 1 μg A317). Serum was collected for antibody analysison days 14 (2wp1), 35 (2wp2) and 49 (4wp2). When measurement of T cellresponses was required, spleens were harvested from 5 mice per group atday 35 or 49 for T cell analysis.

Mouse T Cell Function Assays: Intracellular Cytokines ImmunofluorescenceAssay

Two to five spleens from identically vaccinated BALB/c mice were pooledand single cell suspensions were prepared for culture. Twoantigen-stimulated cultures and two unstimulated cultures wereestablished for each splenocyte pool. Antigen-stimulated culturescontained 1×10⁶ splenocytes, RSV F peptide 85-93 (1×10⁻⁶ M), RSV Fpeptide 249-258 (1×10⁻⁶ M), RSV F peptide 51-66 (1×10⁻⁶ M), anti-CD28mAb (1 mcg/mL), and Brefeldin A (1:1000). Unstimulated cultures did notcontain RSV F peptides, and were otherwise identical to the stimulatedcultures. After culturing for 6 hours at 37° C., cultures were processedfor immunofluorescence. Cells were washed and then stained withfluorecently labeled anti-CD4 and anti-CD8 monoclonal antibodies (mAb).Cells were washed again and then fixed with Cytofix/cytoperm for 20minutes. The fixed cells were then washed with Perm-wash buffer and thenstained with fluorescently labeled mAbs specific for IFN-g, TNF-a, IL-2,and IL-5. Stained cells were washed and then analyzed on an LSR II flowcytometer. FlowJo software was used to analyze the acquired data. TheCD4+8- and CD8+4-T cell subsets were analyzed separately. For eachsubset in a given sample the % cytokine-positive cells was determined.The % RSV F antigen-specific T cells was calculated as the differencebetween the % cytokine-positive cells in the antigen-stimulated culturesand the % cytokine-positive cells in the unstimulated cultures. The 95%confidence limits for the % antigen-specific cells were determined usingstandard methods (Statistical Methods, 7^(th) Edition, G. W. Snedecorand W. G. Cochran).

Mouse T Cell Function Assays: Secreted Cytokines Assay

The cultures for the secreted cytokines assay were similar to those forthe intracellular cytokines immunofluorescence assay except thatBrefeldin A was omitted. Culture supernatants were collected afterovernight culture at 37° C., and were analyzed for multiple cytokinesusing mouse Th1/Th2 cytokine kits from Meso Scale Discovery. The amountof each cytokine per culture was determined from standard curvesproduced using purified, recombinant cytokines supplied by themanufacturer.

2. CNE17 Enhanced Immunogenicity of RSV-F Antigen in a Mouse Model

F-specific serum IgG titers on day 14, 35 and 49 are shown in Tables 14,and 16. RSV serum neutralization titers on day 35 and 49 are shown inTable 17 and T cell responses at day 49 are shown in Table 18 and 19.

TABLE 14 F-specific serum IgG titers of mice at day 14 1 μg 0.1 μg 1 μg10 μg A317 + 1E6 IU A317 CNE17 CNE17 EP VRP 529 2429 3373 5 6041 15302060 4417 88 4912 2734 2012 1927 964 12923 2503 1887 3597 7235 7075 55393174 5731 2558 6829 1033 3904 2852 5105 4885 5110 1481 3739 9806 36801106 2345 4904 2787 9813 1493 3084 3824 2576 8631 3456 2497 3004 18586314 GMT 1980 2398 3590 1180 6685 Serum was collected for antibodyanalysis on days 14 (2wp1). Data are represented as individual animalsand the geometric mean titers of 10 individual mice per group. If anindividual animal had a titer of <25 (limit of detection) it wasassigned a titer of 5.

TABLE 15 F-specific serum IgG titers of mice at day 35 1 μg 0.1 μg 1 μg10 μg A317 + 1E6 IU A317 CNE17 CNE17 EP VRP 958 48079 8473 14612 81304512518 17589 58556 22805 365485 4839 8522 12053 32156 961601 10128 1098520395 24090 349215 18451 30801 51514 31053 297526 9805 13372 26348 18105207652 19154 5137 80686 23918 1580066 4490 47173 21014 9091 900889 1467478232 61076 21006 822285 15223 24135 25499 9835 587121 GMT 8532 2076729111 19117 579033 Serum was collected for antibody analysis on days 35(2wp2). Data are represented as individual animals and the geometricmean titers of 10 individual mice per group. If an individual animal hada titer of <25 (limit of detection) it was assigned a titer of 5.

TABLE 16 F-specific serum IgG titers of mice at day 49 1 μg 0.1 μg 1 μg10 μg A317 + 1E6 IU A317 CNE17 CNE17 EP VRP 958 48079 8473 14612 81304512518 17589 58556 22805 365485 4839 8522 12053 32156 961601 10128 1098520395 24090 349215 18451 30801 51514 31053 297526 9805 13372 26348 18105207652 19154 5137 80686 23918 1580066 4490 47173 21014 9091 900889 1467478232 61076 21006 822285 15223 24135 25499 9835 587121 GMT 8532 2076729111 19117 579033 Serum was collected for antibody analysis on days 49(4wp2). Data are represented as individual animals and the geometricmean titers of 10 individual mice per group. If an individual animal hada titer of <25 (limit of detection) it was assigned a titer of 5.

TABLE 17 RSV serum neutralization titers A317, CNE17, CNE17, VRP 1 μg0.1 μg 1 μg 1E6 IU 2wp2 4wp2 2wp2 4wp2 2wp2 4wp2 2wp2 4wp2 NA <40 NA <40NA <40 265 161 NA <40 NA <40 NA 70 73 64 NA <40 NA <40 NA <40 77 126 NA<40 NA <40 NA 76 140 151 NA <40 NA 42 NA 57 290 194 NA <40 NA 52 NA <40134 123 NA <40 NA <40 NA <40 466 1033 NA <40 NA 173 NA <40 127 174 NA<40 NA <40 NA <40 75 122 NA <40 NA <40 NA <40 77 76 GMT NA <40 NA 29 NA34 139 155 Serum was collected for analysis on days 35 (2wp2) and 49(4wp2). Data are represented as 60% plaque reduction neutralizationtiters of individual mice and the geometric mean titer of 10 individualmice per group. If an individual animal had a titer of <40 (limit ofdetection) it was assigned a titer of 20. NA = not assayed.

TABLE 18 Frequencies of RSV F-specific CD4+ splenic T cells on day 49(4wp2) 4wp2 splenic T CD4 + CD8−: F51-66 peptide restimulation cellresponses IFNg+ IL2+ IL5+ TNFa+ VRP 1E6 IU 0.07 ± 0.06 0.04 ± 0.05 0.00± 0.02 0.10 ± 0.04 1 μg A317 0.00 ± 0.05 0.05 ± 0.04 0.00 ± 0.01 0.03 ±0.02 CNE17, 1 μg 0.00 ± 0.05 0.04 ± 0.04 0.00 ± 0.01 0.05 ± 0.02 CNE17,0.1 μg 0.00 ± 0.05 0.02 ± 0.04 0.00 ± 0.01 0.02 ± 0.02 10 μg A317 + 0.02± 0.06 0.04 ± 0.04 0.01 ± 0.01 0.05 ± 0.03 EP none 0.04 ± 0.06 0.00 ±0.05 0.00 ± 0.02 0.00 ± 0.01 Shown are net (antigen-specific)cytokine-positive frequency (%) ± 95% confidence half-interval. Netfrequencies shown in bold indicate stimulated responses that werestatistically significantly > 0.

TABLE 19 Frequencies of RSV F-specific CD8+ splenic T cells on day 49(4wp2) 4wp2 splenic T CD8 + CD4−: F85-93, F249-258 peptide restimulationcell responses IFNg+ IL2+ IL5+ TNFa+ VRP 1E6 IU 3.48 ± 0.29 1.21 ± 0.18−0.03 ± 0.05 3.31 ± 0.28 1 μg A317 0.74 ± 0.15 0.46 ± 0.11 −0.03 ± 0.040.70 ± 0.14 CNE17, 1 μg 1.25 ± 0.17 0.60 ± 0.12  0.01 ± 0.03 1.15 ± 0.16CNE17, 0.1 μg 0.89 ± 0.15 0.49 ± 0.11 −0.03 ± 0.04 0.83 ± 0.14 10 μgA317 + 0.85 ± 0.15 0.53 ± 0.11  0.01 ± 0.04 0.72 ± 0.15 EP none 0.01 ±0.07 0.00 ± 0.05 −0.02 ± 0.05 0.02 ± 0.06 Shown are net(antigen-specific) cytokine-positive frequency (%) ± 95% confidencehalf-interval. Net frequencies shown in bold indicate stimulatedresponses that were statistically significantly > 0.

As shown in Tables 14-19, CNE17 formulation enhanced immunogenicity, asdetermined by increased F-specific IgG titers (5-fold increase 4wp2),neutralization titers, and CD4 and CD8 T cell responses, relative to thenaked RNA control. Electroporation of RNA enhanced immunogenicityrelative to the naked RNA control, but was lower than CNE17 delivery.Importantly, the immune responses elicited in CNE17 groups fluctuatedmuch less as compared to that of naked RNA. For example, the day 14samples from the 1 μg naked self replicating RNA group gave antibodytiters between 529 and 5110, whereas RNA samples formulated with CNE17at a 1 μg dose gave antibody titers between 1927 and 5731. Additionally,all animals in the CNE17 group responded with a robust response andboosted very well. In contrast, some animals in the naked RNA group thatdid not boost significantly.

Example 5 Immunogenicity of the RNA-Particle Complexes in a Rat Model 1.Methods RSV-F Trimer Subunit Vaccine

The RSV F trimer is a recombinant protein comprising the ectodomain ofRSV F with a deletion of the fusion peptide region preventingassociation with other trimers. The resulting construct forms ahomogeneous trimer, as observed by size exclusion chromatography, andhas an expected phenotype consistent with a postfusion F conformation asobserved by electron microscopy. The protein was expressed in insectcells and purified by virtue of a HIS-tagged in fusion with theconstruct's C-terminus followed by size exclusion chromatography usingconventional techniques. The resulting protein sample exhibits greaterthan 95% purity. For the in vivo evaluation of the F-subunit vaccine,100 μg/mL trimer protein was adsorbed on 2 mg/mL alum using 10 mMHistidine buffer, pH 6.3 and isotonicity adjusted with sodium chlorideto 150 mM. F-subunit protein was adsorbed on alum overnight with gentlestirring at 2-8° C.

Vaccination and Challenge of Cotton Rats

Female cotton rats (Sigmodon hispidis) were obtained from HarlanLaboratories. All studies were approved and performed according toNovartis Animal Care and Use Committee. Groups of animals were immunizedintramuscularly (i.m., 100 μl) with the indicated vaccines on days 0 and21. Serum samples were collected 3 weeks after the first immunizationand 2 weeks after the second immunization Immunized or unvaccinatedcontrol animals were challenged intranasally (i.n.) with 1×10⁵ PFU RSV 4weeks after the final immunization. Blood collection and RSV challengewere performed under anesthesia with 3% isoflurane using a precisionvaporizer.

RSV F-specific ELISA

Individual serum samples were assayed for the presence of RSV F-specificIgG by enzyme-linked immunosorbent assay (ELISA). ELISA plates (MaxiSorp96-well, Nunc) were coated overnight at 4° C. with 1 μg/ml purified RSVF (delp23-furdel-trunc uncleaved) in PBS. After washing (PBS with 0.1%Tween-20), plates were blocked with Superblock Blocking Buffer in PBS(Thermo Scientific) for at least 1.5 hr at 37° C. The plates were thenwashed, serial dilutions of serum in assay diluent (PBS with 0.1%Tween-20 and 5% goat serum) from experimental or control cotton ratswere added, and plates were incubated for 2 hr at 37° C. After washing,plates were incubated with horse radish peroxidase (HRP)-conjugatedchicken anti-cotton rat IgG (Immunology Consultants Laboratory, Inc,diluted 1:5,000 in assay diluent) for 1 hr at 37° C. Finally, plateswere washed and 100 μl of TMB peroxidase substrate solution (Kirkegaard& Perry Laboratories, Inc) was added to each well. Reactions werestopped by addition of 100 μl of 1M H₃PO₄, and absorbance was read at450 nm using a plate reader. For each serum sample, a plot of opticaldensity (OD) versus logarithm of the reciprocal serum dilution wasgenerated by nonlinear regression (GraphPad Prism). Titers were definedas the reciprocal serum dilution at an OD of approximately 0.5(normalized to a standard, pooled sera from RSV-infected cotton ratswith a defined titer of 1:2500, that was included on every plate).

Micro Neutralization Assay

Serum samples were tested for the presence of neutralizing antibodies bya plaque reduction neutralization test (PRNT). Two-fold serial dilutionsof HI-serum (in PBS with 5% HI-FBS) were added to an equal volume of RSVLong previously titered to give approximately 115 PFU/25 μl. Serum/virusmixtures were incubated for 2 hours at 37° C. and 5% CO2, to allow virusneutralization to occur, and then 25 μl of this mixture (containingapproximately 115 PFU) was inoculated on duplicate wells of HEp-2 cellsin 96 well plates. After 2 hr at 37° C. and 5% CO2, the cells wereoverlayed with 0.75% Methyl Cellulose/EMEM 5% HI-FBS and incubated for42 hours. The number of infectious virus particles was determined bydetection of syncytia formation by immunostaining followed by automatedcounting. The neutralization titer is defined as the reciprocal of theserum dilution producing at least a 60% reduction in number of synctiaper well, relative to controls (no serum).

Viral Load

Viral load in the lung was determined by plaque assay. Specifically,lungs were harvested 5 days post RSV infection and one right lobe wasplaced into 2.5 ml Dulbecco's Modified Eagle Medium (DMEM, Invitrogen)with 25% sucrose and disrupted with a tissue homogenizer. Cell-freesupernatants from these samples were stored at −80° C. To assay forinfectious virus, dilutions of clarified lung homogenate (in PBS with 5%heat-inactivated fetal bovine serum, HI-FBS) were inoculated onconfluent HEp-2 cell monolayers in a volume of 200 μl/well of a 12-wellplate. After 2 hrs with periodic gentle rocking (37° C., 5% CO₂), theinoculum was removed, and cells were overlaid with 1.5 ml of 1.25%SeaPlaque agarose (Lonza) in Eagle's Minimal Essential Medium (EMEM,Lonza) supplemented with 5% HI-FBS, glutamine, and antibiotics. After3-4 days of incubation, cells were again overlaid with 1 ml of 1.25%agarose in EMEM (Sigma) containing 0.1% neutral red (Sigma). Plaques arecounted one day later with the aid of a light box.

Cotton Rat Lung Pathology

Five days after RSV challenge lungs were harvested and 4 lobes from eachanimal were collected and fixed with 10% neutral buffered formalin (NBF)by gentle intratracheal instillation followed by immersion fixation.Tissues were processed routinely to prepare hematoxylin & eosin-stainedsections for microscopic examination. Findings were evaluated using amodification of previously published criteria [Prince G A, et al., 2001]for the following parameters: peribronchiolitis, alveolitis, bronchitis,perivascular cellular infiltrates, and interstitial pneumonitis. Lesionswere graded on a 4-point semiquantitative scale. Minimal (+) changecontained one or a few small foci; mild (++) change was composed ofsmall- to medium-size foci; moderate (+++) change contained frequentand/or moderately-sized foci; and marked (++++) change showed extensiveto confluent foci affecting most/all of the tissue.

2. Cotton Rat RSV Challenge Study

A317 replicon, which expresses the surface fusion glycoprotein of RSV(RSV-F) was used for this study. Cotton rats (Sigmodon hispidus), 8animals per group, were given bilateral intramuscular vaccinations (50μL per leg) on days 0 and 21 with naked self-replicating RNA (A317, 1 μgor 10 μg), self-replicating RNA formulated with CNE17 (A317, 0.1 μg or 1μg), VRPs (5×10⁶ IU) expressing RSV-F, F-trimer/alum subunit (10 μg), orformalin inactivated RSV vaccine (5200 FI-pfu). Serum was collected forantibody analysis on days 14 (2wp1) and 35 (2wp2). All animals werechallenged with 1×10⁵ pfu RSV intranasally on day 49 and lungs werecollected on day 54 (5dpc) for determination of viral load and lungpathology.

F-specific serum IgG titers on day 14 and 35 are shown in Table 20;individual antibody titers for 8 animals from selected groups at 2wp2are shown in Table 21; RSV serum neutralization titers on days 14 and 35are shown in Table 22; lung viral titers 5 days post RSV challenge areshown in Table 23; and Lung alveolitis scores 5 days post RSV challengeare shown in Table 24.

TABLE 20 F-specific serum IgG titers of cotton rats (Sigmodon hispidus)F-specific IgG F-specific IgG vaccine dose 2wp1 2wp2 Naked A317 10 μg198 1599 Naked A317  1 μg 78 526 CNE17  1 μg 408 4918 CNE17 0.1 μg  3252512 VRP 5 × 106 IU 961 5864 F-trimer/alum 10 μg 3526 111893 FI-RSV 5200FI-pfu 17 2074 none 5 5 8 animals per group, after intramuscularvaccinations on days 0 and 21. Serum was collected for antibody analysison days 14 (2wp1) and 35 (2wp2), all animals were challenged with 1 ×10⁵ pfu RSV intranasally on day 49. Lungs were collected on day 54(5dpc) for determination of viral load and lung pathology. Data arerepresented as geometric mean titers of 8 individual cotton rats pergroup. If an individual animal had a titer of <25 (limit of detection)it was assigned a titer of 5.

TABLE 21 Individual antibody titers at 2wp2 10 μg 1 μg 0.1 μg 1 μg A317A317 CNE17 CNE17 1778 612 3967 3740 1534 409 2360 3199 3144 1039 17863998 1174 116 3097 7173 1719 1086 1075 9005 488 869 2956 6170 1586 7421496 6406 3200 276 6431 2800 Individual antibody titers for 8 animalsfrom selected groups (naked RNA and CNE formulated RNA).

TABLE 22 RSV serum neutralization titers of cotton rats (Sigmodonhispidus) PRNT60 PRNT60 vaccine dose 2wp1 2wp2 Naked A317 10 μg 78 240Naked A317  1 μg 58 70 CNE17  1 μg 91 269 CNE17 0.1 μg  63 145 VRP 5 ×10⁶ IU 149 683 F-trimer/alum 10 μg 142 >5120 FI-RSV 5200 FI-pfu 28 38none 30 <20 8 animals per group, after intramuscular vaccinations ondays 0 and 21. Serum was collected for analysis on days 14 (2wp1) and 35(2wp2). Data are represented as 60% plaque reduction neutralizationtiters. Geometric mean titer of 2 pools of 4 cotton rats per group. Ifan individual animal had a titer of <25 (limit of detection) it wasassigned a titer of 5.

TABLE 23 Lung viral titers 5 days post RSV challenge of cotton rats(Sigmodon hispidus) vaccine dose pfu/g lung 5dpc Naked A317 10 μg 397Naked A317  1 μg 659 CNE17  1 μg 414 CNE17 0.1 μg  572 VRP 5 × 106 IU359 F-trimer/alum 10 μg 190 FI-RSV 5200 FI-pfu 5248 8 animals per group,after intramuscular vaccinations on days 0 and 21. Serum was collectedfor analysis on days 14 (2wp1) and 35 (2wp2). Data are represented as60% plaque reduction neutralization titers. Geometric mean titer of 2pools of 4 cotton rats per group. If an individual animal had a titer of<25 (limit of detection) it was assigned a titer of 5.

TABLE 24 Lung alveolitis 5 days post RSV challenge of cotton rats(Sigmodon hispidus) # of cotton rats with indicated alveolitis scorevaccine dose 0 1 2 3 4 Naked A317 10 μg 8 Naked A317 1 μg 8 CNE17 1 μg 8CNE17 0.1 μg 7 1 VRP 5 × 10⁶ IU 3 4 1 F-trimer/alum 10 μg 7 1 FI-RSV5200 FI-pfu 1 4 3 none (challenged) 5 3 8 animals per group, afterintramuscular vaccinations on days 0 and 21. All animals were challengedwith 1 × 10⁵ pfu RSV intranasally on day 49. Lungs were collected on day54 (5dpc) for determination of viral load and lung pathology. Lesionswere graded on a 4-point semiquantitative scale. Minimal (1) changecontained one or a few small foci; mild (2) change was composed ofsmall-to medium-size foci; moderate (3) change contained frequent and/ormoderately-sized foci; and marked (4) change showed extensive toconfluent foci affecting most/all of the tissue.

This study shows the immunogenicity and protective capacity of repliconRNA in the cotton rat RSV model. Unformulated replicon RNA induced serumF-specific IgG and RSV neutralizing antibodies after one vaccination,and that these responses were boosted by a second vaccination. CNE waseffective in this model, boosting F-specific IgG titers to 1 μg repliconRNA approximately 9-fold and neutralization titers by 4-fold after thesecond vaccination. Additionally, CNE17 reduced the considerablevariations of the immune responses that were observed when naked RNA wasused, regardless of the doses (0.1 or 1 μg), and all animals respondedto vaccination. All replicon RNA vaccines provided protection from anasal RSV challenge, reducing the lung viral load 5 days post RSVchallenge more than 3 orders of magnitude. The magnitude and protectivecapacity of the immune response generated by 1 μg replicon RNAformulated with CNE was within 2-fold the response elicited by 5×10⁶VRPs.

Example 6 The Effect of Particle Size on Immunogenicity

This example shows that particle size affects the immunogenicity of theCNE/RNA formulations.

Protocols for particle size assay and in vivo SEAP assay are describedin Example 2. Protocols for murine immunogenicity studies are describedin Example 3.

FIG. 8A shows the results (arithmetic mean) of the in vivo SEAP assay.FIG. 8B shows the total IgG titers of individual animals in the BALB/cmice at 2wp1 and 2wp2 time points.

RNA complexation with CNE17 increased particle size from about 220 nm toabout 300 nm (data not shown). As shown in FIGS. 8A and 8B, as particlesize increased, the expression levels of SEAP were reduced, and the hostimmune responses were also decreased.

Example 7 Assessing the Effects of Alternative Cationic Lipids onImmunogenicity 1. Materials and Methods. Preparation of CNEs

A series of emulsions were made using the following cationic lipids:DLinDMA, DOTMA, DOEPC, DSTAP, DODAC, and DODAP. Table 25 describes thecomponents of the emulsions.

CNEs were prepared according the protocols described in Example 1. TheRNA/CNE complex were prepared according the protocols described inExample 2.

TABLE 25 Cationic mg/ml CNE Lipid (+) +Lipid Surfactant SqualeneBuffer/water CMF20 DLinDMA 1.25 0.5% SPAN 85 4.3% 10 mM citrate bufferpH 6.5 0.5% Tween 80 (in RNase-free dH₂O, no DCM) CMF21 DLinDMA 1.250.5% SPAN 85 4.3% 10 mM citrate buffer pH 6.5 0.5% Tween 80 (inRNase-free dH₂O, & 50° C. heat & sonication to solubilize; solventevaporated post 1st homogenization) CMF36 DODAP 1.3 0.5% SPAN 85 4.3% 10mM citrate buffer pH 6.5 0.5% Tween 80 (in RNase-free dH₂O, CHCl₃;solvent evaporated prior to homogenization) CMF37 DOTMA 1.35 0.5% SPAN85 4.3% 10 mM citrate buffer pH 6.5 0.5% Tween 80 (in RNase-free dH₂O,no DCM) CMF38 DOEPC 1.7 0.5% SPAN 85 4.3% 10 mM citrate buffer pH 6.50.5% Tween 80 (in RNase-free dH₂O, no DCM) CMF39 DDA 1.65 0.5% SPAN 854.3% 10 mM citrate buffer pH 6.5 0.5% Tween 80 (in RNase-free dH₂O,solvent evaporated post 1st homogenization) CMF42 DSTAP 1.4 0.5% SPAN 854.3% 10 mM citrate buffer pH 6.5 0.5% Tween 80 (in RNase-free dH₂O, DCMand methanol; solvents evaporated prior to homogenization) CMF43 DODAC1.17 0.5% SPAN 85 4.3% 10 mM citrate buffer pH 6.5 0.5% Tween 80 (inRNase-free dH₂O, no DCM)

Murine Immunogenicity Studies

The emulsions were tested at 10:1 N/P, 12:1 N/P or 18:1 N/P ratios (seeTable 26). Then RNA replicon and the emulsions were complexed aspreviously described in Example 2. BALB/c mice, 5-10 animals per group,were given bilateral intramuscular vaccinations (50 μL per leg) on days0 with naked self-replicating RNA (A317, 1 μg), RV01(15) (1 μg of A317formulated in a liposome that contained 40% D11n DMA, 10% DSPC, 48%Chol, 2% PEG DMG 2000), self-replicating RNA (A317, 1 μg) formulatedwith CNE13, CNE17, CMF37, CMF38, or CMF42.

2. CNE-formulated RNA Enhanced Immunogenicity of RSV-F Antigen in aMouse Model

Total serum IgG titers (Geometric Mean Titers) from the groups of BALB/cmice on day 14 and 35 are shown in Table 26 (groups 1-8). CMF37(DOTMA)-formulated RNA enhanced host immune response well, and the IgGtiters were comparable to that CNE17 (DOTAP). CMF38 (DOEPC)-formulatedRNA elicited a slightly higher IgG titer than that of CNE17, but theenhancement was not statistically significant. DSTAP-formulated RNA didnot significantly enhance host immune response, and the low IgG titerswere likely due to the low solubility of DSTAP in squalene.CNE13-formulated RNA enhanced IgG titers about 1.5-fold greater thanthat of liposome (DDA)-formulated RNA. Total antibody titers induced byCMF43 (DODAC)-formulated RNA were lower than that of CNE17 (Table 28,Groups 7 and 8).

TABLE 26 Group Description N:P 2wp2/2wp1 # Emulsion ratio 2wp1 2wp2ratio 1 1 ug vA317 — 77 1,710 22.2 2 RV01(15) — 3,441 59,557 17.3 3CNE17 DOTAP 10:1 1,474 6,512 4.4 4 CNE13 DDA 18:1 482 8,385 17.4 5 CMF37DOTMA 10:1 474 6,556 13.8 6 CNE16 DOEPC 12:1 1,145 9,673 8.4 7 CMF42DSTAP 10:1 22 148 6.7 8 DDA Liposomes 18:1 898 5,333 5.9 9 CNE17 with300 mM 10:1 1,807 6,445 3.6 Trehalose 10 CNE17 with 300 mM 10:1 1,0425,515 5.3 Sucrose 11 CNE17 with 300 mM 10:1 1,209 8,874 7.3 Sorbitol 12CNE17 with 300 mM 10:1 1,247 7,956 6.4 Dextrose Groups 1-8 had 5animals/group, and groups 9-12 had 10 animals/group.

Example 8 Assessing the Effects of Buffer Compositions on Immunogenicity

In this example, various emulsions based on CNE17 but with differentbuffer components were prepared. Table 27 shows the compositions of thebuffer-modified emulsions.

TABLE 27 Base Emulsion Buffer/water CNE17: 4.3% Squalene, 0.5% SPAN 85,0 mM citrate buffer (in 0.5% Tween 80, 1.4 mg/ml DOTAP RNase-free dH₂O,no DCM) CNE17: 4.3% Squalene, 0.5% SPAN 85, 1 mM citrate buffer (in 0.5%Tween 80, 1.4 mg/ml DOTAP RNase-free dH₂O, no DCM) CNE17: 4.3% Squalene,0.5% SPAN 85, 5 mM citrate buffer (in 0.5% Tween 80, 1.4 mg/ml DOTAPRNase-free dH₂O, no DCM) CNE17: 4.3% Squalene, 0.5% SPAN 85, 10 mMcitrate buffer pH 6.5 0.5% Tween 80, 1.4 mg/ml DOTAP 300 mM TrehaloseCNE17: 4.3% Squalene, 0.5% SPAN 85, 10 mM citrate buffer pH 6.5 0.5%Tween 80, 1.4 mg/ml DOTAP 300 mM Sucrose CNE17: 4.3% Squalene, 0.5% SPAN85, 10 mM citrate buffer pH 6.5 0.5% Tween 80, 1.4 mg/ml DOTAP 300 mMSorbitol CNE17: 4.3% Squalene, 0.5% SPAN 85, 10 mM citrate buffer pH 6.50.5% Tween 80, 1.4 mg/ml DOTAP 300 mM Dextrose

In vitro binding assay showed that reducing the concentration of citratebuffer caused RNA to bind more tightly (data not shown).

Results from murine immunogenicity studies showed that adding sugars toCNE17 did not significantly impact the immunogenicity of theCNE17-formulated RNA (Table 26, groups 9-12)). Slight increases in IgGtiters were observed with the addition of sorbitol and dextrose.

Table 28 summarizes the results of murine immunogenicity studies whenCNE17-formulated RNAs were prepared using different buffer systems.

TABLE 28 Description 2wp2/2wp1 Group # RNA Emulsion N:P ratio 2wp1 2wp2ratio 1 1 μg PBS — 100 2269 23 RSV-F* 2 RV01(15) PBS — 8388 105949 13 31 μg CNE17 with 280 mM 10:1 898 9384 10 RSV-F* Sucrose 4 1 μg CNE17 with280 mM 10:1 1032 3184 3.1 RSV-F** sucrose, 10 mM NaCl, 1 mM Citrate, 5CNE17 with 280 mM 10:1 79 895 11.3 sucrose, 10 mM NaCl, 1 mM Citrate,0.5% (w/v) and Pluronic F127 *vA375 replicon, **vA317 replicon.Replicons were Ambion transcribed in HEPES buffer, then (i) LiClprecipitated, (ii) capped in Tris buffer, and (iii) LiCl precipitated.All groups had 8 animals/group.

Different buffer compositions also affected particle size. As shown inFIG. 9, addition of sugar (sucrose) decreased the particle size of theRNA/CNE complex (FIG. 9A); addition of low concentrations of NaCl (at 10mM) also decreased the particle size of the RNA/CNE complex (FIG. 9A).Citrate buffer did not affect the particle size of the RNA/CNE complex(FIG. 9B).

The effects of polymers on particle size are shown in FIG. 9C. Inparticular, addition of 0.5% pleuronic F127 to RNA buffer reduced theparticle size of the RNA/CNE complex to the pre-complexation size (CNEparticles without RNA).

The total antibody titers and neutralizing antibody titers of CNE17 inpreferred buffer systems, 280 mM sucrose, 10 mM NaCl, and 1 mM Citrate;or 280 mM sucrose, 10 mM NaCl, 1 mM Citrate, and 0.5% (w/v) PluronicF127, are shown in Table 28 (groups 4 and 5).

Example 9 Assessing the Effects of Peg-Lipids on Immunogenicity

In this example, a series of emulsions were made using PEG-lipids. Table29 shows the compositions of these PEG-lipid based emulsions.

TABLE 29 Cationic mg/ml + CNE Lipid (+) Lipid PEG-lipid SqualeneBuffer/water CMF22 DOTAP 1.4 PEG2K C18-1 4.3% 10 mM citrate buffer pH6.5 10 mg/mL (in RNase-free dH₂O) CMF23 DOTAP 1.4 PEG2K C18-1 4.3% 10 mMcitrate buffer pH 6.5 5 mg/mL (in RNase-free dH₂O) CMF24 DOTAP 1.4 PEG2KC14 4.3% 10 mM citrate buffer pH 6.5 9.6 mg/mL (in RNase-free dH₂O)CMF25 DOTAP 1.4 PEG2K C141 4.3% 10 mM citrate buffer pH 6.5 9.25 mg/mL(in RNase-free dH₂O) CMF26 DOTAP 1.4 PEG2K C18-1 4.3% 10 mM citratebuffer pH 6.5 0.7 mg/mL (in RNase-free dH₂O) CMF27 DOTAP 1.4 PEG2K C18-14.3% 10 mM citrate buffer pH 6.5 1.4 mg/mL (in RNase-free dH₂O) CMF28DOTAP 1.4 PEG2K C14 4.3% 10 mM citrate buffer pH 6.5 0.7 mg/mL (inRNase-free dH₂O) CMF29 DOTAP 1.4 PEG2K C14 4.3% 10 mM citrate buffer pH6.5 1.4 mg/mL (in RNase-free dH₂O)

For all of the emulsion, a stock solution of 10 mg/mL DOTAP in DCM wereused, and the solvent was evaporated after the 1st homogenization.Murine immunogenicity studies were carried out as described above inExample 7.

Table 30 shows the pooled antibody titers at the 2wp1 and 4wp2 timepoints. For the CNE13 group, the average of individual animal titers,and the geo mean titers are shown. As shown in Table 30, emulsions madewith PEG-lipids were effective in inducing immune response against theRSV-F antigen, but the total antibody titers were at a lower level ascompared to CNE17-formulated RNA. In addition, increasing theconcentration of the PEG-lipids led to a decrease in antibody titers.

TABLE 30 2wp1 pooled 4wp2 pooled Group RNA Formulation titer titer 1 1μg none 780 2794 2 RSV-F* CNE17 (10:1 N/P ratio) 1,783 12907 3 CMF26(6:1 N/P ratio), 323 4661 (0.7 mg/mL 2K PEG C18-1) 4 CMF26 (10:1 N/Pratio), 336 6588 (0.7 mg/mL 2K PEG C18-1) 5 CMF27 (6:1 N/P ratio), 2092119 (1.4 mg/mL 2K PEG C18-1) 6 CMF27 (10:1 N/P ratio), 525 3770 (1.4mg/mL 2K PEG C18-1) 7 CMF28 (6:1 N/P ratio), 906 6923 (0.7 mg/mL 2K PEGC14) 8 CMF28 (10:1 N/P ratio), 1,280 5532 (0.7 mg/mL 2K PEG C14) 9 CMF29(6:1 N/P ratio), 159 1603 (1.4 mg/mL 2K PEG C14) 10 CMF29 (10:1 N/Pratio), 110 4041 (1.4 mg/mL 2K PEG C14) 11 CNE13 (18:1 N/P ratio) 3,026(average); 25,738 (average); 2891 (GMT) 23068 (GMT) *vA317 replicon,Groups 1-10 had 5 animals/group and group 11 had 10 animals/group.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments.

1. A composition comprising an RNA molecule complexed with a particle ofa cationic oil-in-water emulsion, wherein the particle comprises (a) anoil core that is in liquid phase at 25° C., and (b) a cationic lipid. 2.The composition of claim 1, wherein the particle further comprises asurfactant.
 3. The composition of claim 2, wherein the surfactant is anonionic surfactant.
 4. The composition of claim 3, wherein thesurfactant is SPAN85 (sorbtian trioleate), TWEEN 80 (polysorbate 80), ora combination thereof.
 5. The composition of claim 3, wherein thecationic oil-in-water emulsion comprises from about 0.01% to about 2.5%(v/v) nonionic surfactant.
 6. The composition of claim 1, wherein thecomposition comprises from about 0.2% to about 9% (v/v) of said oil. 7.The composition of claim 1, wherein the oil core comprises Soybean oil,Sunflower oil, Olive oil, Squalene, Squalane or combinations thereof. 8.The composition of claim 1, wherein the cationic lipid is selected fromthe group consisting of: 1,2-dioleoyloxy-3-(trimethylammonio)propane(DOTAP), 3β-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), and1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA).
 9. The compositionof claim 1, wherein the cationic lipid is1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP).
 10. The compositionof claim 9, wherein the cationic oil-in-water emulsion comprises fromabout 0.5 mg/ml to about 5 mg/ml DOTAP.
 11. The composition of claim 1,wherein the cationic lipid is3β-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DC Cholesterol)or dimethyldioctadecylammonium (DDA).
 12. The composition of claim 11,wherein the composition comprises from about 0.1 mg/ml to about 5 mg/mlDC Cholesterol or DDA.
 13. The composition of claim 1, wherein thecationic lipid is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTMA), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC).
 14. The compositionof claim 13, wherein the composition comprises from about 0.5 mg/ml toabout 5 mg/ml DOTMA, DOEPC, or DODAC.
 15. The composition of claim 1,wherein the cationic oil-in-water emulsion further comprises a polymeror a surfactant in the aqueous phase of the emulsion.
 16. Thecomposition of claim 15, wherein the polymer is a poloxamer.
 17. Thecomposition of claim 16, wherein the cationic oil-in-water emulsioncomprises from about 0.1% to about 10% (v/v) polymer.
 18. Thecomposition of claim 1, wherein the RNA molecule is a self-replicatingRNA molecule that encodes an antigen.
 19. The composition of claim 1,wherein the average diameter of the emulsion particles is no greaterthan 200 nm.
 20. The composition of claim 1, wherein the N/P ratio ofthe emulsion is from 4:1 to 14:1.
 21. The composition of claim 1,wherein the average diameter of the emulsion particles is from about 80nm to about 180 nm, and the N/P ratio of the emulsion is at least 4:1.22. The composition of claim 1, wherein the composition is buffered andhas a pH of about 6.0 to about 8.0.
 23. The composition of claim 22,wherein the pH is about 6.2 to about 6.8.
 24. The composition of claim21, wherein the composition further comprises an inorganic salt, and theconcentration of inorganic salt is no greater than 30 mM.
 25. Thecomposition of claim 1, wherein the composition further comprises anonionic tonicifying agent.
 26. The composition of claim 25, wherein thenonionic tonicifying agent is selected from the group consisting of asugar, a sugar alcohol and combinations thereof.
 27. The composition ofclaim 26, wherein the nonionic tonicifying agent is present in aconcentration of about 280 mM to about 300 mM
 28. The composition ofclaim 25, wherein the composition is isotonic.
 29. A method ofgenerating an immune response in a subject, comprising administering toa subject in need thereof a therapeutically effective amount of thecomposition of claim
 1. 30. A composition comprising a negativelycharged molecule complexed with a particle of a cationic oil-in-wateremulsion, wherein the particle comprises (a) an oil core, (b) a cationiclipid, and (c) a phospholipid.
 31. A method of generating an immuneresponse in a subject, comprising administering to a subject in needthereof a therapeutically effective amount of the composition of claim30.
 32. A method of preparing a composition that comprises a negativelycharged molecule complexed with a particle of a cationic oil-in-wateremulsion, comprising providing (a) a cationic oil-in-water emulsion,wherein the emulsion particles comprises (i) an oil core that is inliquid phase at 25° C., and (ii) a cationic lipid, and optionally (iii)a phospholipid; and (b) an aqueous solution comprising the negativelycharged molecule; and combining (a) and (b) such that the negativelycharged molecule complexes with a particle of the cationic oil-in-wateremulsion.
 33. The method of claim 32, wherein the negatively chargedmolecule is a nucleic acid molecule that encodes an antigen.
 34. Themethod of claim 33, wherein the nucleic acid molecule is an RNAmolecule.
 35. The method of claim 34, wherein the RNA molecule is aself-replicating RNA molecule.
 36. The method of claim 32, wherein thecationic oil-in-water emulsion and the RNA solution are combined atabout 1:1 (v/v) ratio.